Curcumin suppresses tumorigenesis by ferroptosis in breast cancer

Xuelei Cao; Yao Li; Yongbin Wang; Tao Yu; Chao Zhu; Xuezhi Zhang; Jialiang Guan

doi:10.1371/journal.pone.0261370

Peer Review History

Original SubmissionJune 23, 2021
11 Aug 2021 Decision Letter - Irina V. Balalaeva, Editor PONE-D-21-20659 Curcumin suppresses tumorigenesis by ferroptosis in breast cancer PLOS ONE Dear Dr. Guan, 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, pay attention, that The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception. 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. 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Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Yes Reviewer #3: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: Yes Reviewer #3: I Don't Know ****** 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: No Reviewer #2: Yes Reviewer #3: 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: No Reviewer #2: Yes Reviewer #3: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Although the authors showed that curcumin suppressed viability of breast cancer cells and tumor growth at relatively high concentrations, it is also toxic to non-tumor cells such as mouse hippocampal HT22, PC12 and NIH-3T3 cells (Hirata et al., ACS Chem Neurosci 11, 76-85, 2020; Meng et al., PLOS ONE 9, e85570, 2014; Zhang et al., Mol Immunol 116, 191-198, 2019). Therefore, the cytotoxic effect of curcumin is not specific for breast cancer cells. Line 61-62. This sentence is inaccurate, or misleading information. Lin et al. analyzed a cohort of 79,335 patients with breast cancer between 2000 and 2010 and showed that danshen improves survival of breast cancer patients. They also showed that dihydroisotanshinone I (DT), a pure compound present in danshen, inhibits the growth of breast carcinoma cells, including MCF-7 and MDA-MB-231 and induces both apoptosis and ferroptosis in those cultured cells. DT-induced ferroptosis is characterized by reducing GSH content and GPX4 protein, and increasing lipid peroxidation. Therefore, there is no direct evidence that danshen prolonged the survival of patients with breast cancer by inducing ferroptosis. Figure 2D, Figure 3H: 1. According to these results, curcumin increased ferrous ion levels in MCF-7 and MDA-MB-453 cells. However, curcumin has effective ferrous ions chelating capacity (Ak and Gulcin, Chemico-Biol Int 174, 27-37, 2008). In fact, curcumin reduces ferrous ions in cultured cells (Hirata et al., ACS Chem Neurosci 11, 76-85, 2020) and in in vitro assay (Shome et al., Biotechnology and Applied Biochemistry 68, 603-615 Biotechnology and Applied Biochemistry, 2021) at concentrations similar to those reported in this study. The authors need to examine the effect defferoxamine on ferrous ion levels as a positive control. 2. Please check whether the unit of ferrous ion is correct. 3. The relative of intracellular Fe2+ expression …: Intracellular Fe2+ level in both … Minor points: Figure 1: Is the concentration of erastin 10 nM? Figure legends: The number of samples used in the experiments and statistics needs to be clarified. Reviewer #2: In this article, the authors performed a series of in vitro and in vivo experiments to find out novel anti-tumorigenic mechanistic insight of curcumin. And they revealed that curcumin administration induced ferroptosis by enhancing the levels of lipid reactive oxygen species (ROS), malondialdehyde (MDA) of lipid peroxidation, and intracellular Fe2+ level in BC cells. Furthermore, treatment with curcumin significantly suppresses tumorigenesis in BC via upregulating SLC1A5 expression, which is an essential transporter for glutamine uptake. As a whole, the authors performed thorough experimental plan, and their finding are quite interesting. However, the authors need further improvement of this article to grown-up to publishing-quality. The followings are the issues that the authors should be addressed. #Because there are some other published article in which Li R, et al, revealed the anti-tumorigenic effect of curcumin by altering other ferroptosis associated gene (HO-1) in breast cancer area (Oxid Med Cell Longev. 2020 Nov 18;2020:3469840), the authors should add this article as reference and should add some discussion with regard to this article. #As for Figure2A,2B, and 3F, please add the title of X-axis. Lipid ROS level? #How about changing the position of Figure 5F to 5A in order to explain the less-toxic effect of curcumin to mice? Reviewer #3: In this article, the authors show that curcumin exhibits anti-tumorigenesis activity in breast cancer cell lines by promoting SLC1A5-mediated ferroptosis (lipid ROS, lipid peroxidation end-product MDA accumulation, and intracellular Fe2+ levels). Objectives are clear, experiments are well designed, and methodology is adequate to obtain the results. The article is well written MAJOR ISSUES 1) Which diluent is used to dissolve curcumin? It must be checked that the vehicle does not reduce cells viability. 2) Authors explain the role of ASCT2, SLC1A5 and SCL38A1 in glutamine uptake, but they only measured SLC1A5. Then they argue about the role of SLC1A5 in ferroptosis induced by curcumin but not about other receptors. Other receptors should be measured to confirm SLC1A5 implication. 3) Inmunohistochemistry staining should be quantified; a representative image is not enough to conclude Ki-67 and SLC1A5 increase. 4) To confirm the role of ferroptosis in vivo, authors should perform the same experiment but treating mice with ferrostatin and other cell death inhibitors, such as ZVAD and NS. In addition, authors should measure other ferroptosis markers than just MDA in the tumor to conclude the role of this pathway. 5) In the discussion sections, authors report that curcumin promotes ferroptosis. However, there are contradictory data in the bibliography about this issue, see reference 38 (Guerrero-Hue et al.). Authors should discuss about the possible dual role of curcumin on ferroptosis. 6) Authors should add in the figure legend the time in which each experiment was performed. ****** 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: No Reviewer #3: Yes:** Juan Antonio Moreno [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.0261370.r001
Revision 1
29 Sep 2021 Author Response RE: Manuscript ID: PONE-D-21-20659 Dear Dr. Balalaeva, We are indeed very grateful to the careful and thoughtful comments of three reviewers and your suggestions on our manuscript entitled “Curcumin suppresses tumorigenesis by ferroptosis in breast cancer” by Cao X et al. Based on the suggestions from you and the reviewers, we have carried out some additional experiments and revised the manuscript. In this letter, we have listed our responses to the specific comments/questions raised by each reviewer, and incorporated all necessary changes in the revision. Editor: 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. Response: We revised our manuscript to meet PLOS ONE's style requirements. 2. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability. Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized. Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover letter. Response: We updated the Data Availability statement in this revision by stating that “All relevant data are within the paper and its Supporting information files”. 3. 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. Response: Raw images of Western blot from Figs 3 and 4 were provided as S1 Raw images in this revision. 4. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ Response: An ORCID iD for the corresponding author was updated in this submission. Again, we really appreciate the reviewer’s careful reading and suggestions on our manuscript, and thank you very much for your consideration of our paper for publication in Plos one. Sincerely yours, Jialiang Guan, The Department of Emergency Internal Medicine, The Affiliated Hospital of Qingdao University, E-mail: gjlqdfy@126.com Reviewer #1: Question #1: Although the authors showed that curcumin suppressed viability of breast cancer cells and tumor growth at relatively high concentrations, it is also toxic to non-tumor cells such as mouse hippocampal HT22, PC12 and NIH-3T3 cells (Hirata et al., ACS Chem Neurosci 11, 76-85, 2020; Meng et al., PLOS ONE 9, e85570, 2014; Zhang et al., Mol Immunol 116, 191-198, 2019). Therefore, the cytotoxic effect of curcumin is not specific for breast cancer cells. Response #1: Thanks for your nice comments on our article. Recently, several studies have demonstrated that curcumin had anticancer and chemoprevention effects on breast cancer through inhibiting cancer proliferation [1] and tumor metastasis [2], as well as enhancing the sensitivity of breast cancer cells to chemotherapeutic drugs [3-5]. Moreover, our data showed that 2-50 μM curcumin significantly reduced the viabilities of breast cancer cell lines (MDA-MB-453 and MCF-7). Therefore, the above results indicated the curcumin serves as an adjunct therapy in breast cancer. Question #2: Line 61-62. This sentence is inaccurate, or misleading information. Response #2: We rewrote this sentence as “It was reported that danshen, a traditional Chinese medicine, improved survival of patients with breast cancer and induced ferroptosis and apoptosis of breast cancer cells”. (Line 62-63, page 3 in this revision.) Question #3: Lin et al. analyzed a cohort of 79,335 patients with breast cancer between 2000 and 2010 and showed that danshen improves survival of breast cancer patients. They also showed that dihydroisotanshinone I (DT), a pure compound present in danshen, inhibits the growth of breast carcinoma cells, including MCF-7 and MDA-MB-231 and induces both apoptosis and ferroptosis in those cultured cells. DT-induced ferroptosis is characterized by reducing GSH content and GPX4 protein, and increasing lipid peroxidation. Therefore, there is no direct evidence that danshen prolonged the survival of patients with breast cancer by inducing ferroptosis. Response #3: In this study, the results showed that the use of Danshen ≥84 g was highly associated with decreased mortality (the adjusted HR of Danshen ≥84 g users was 0.54 [95% CI, 0.46–0.63] (p <0.001). Moreover, the use of Danshen for >28 days remained highly associated with decreased mortality (the adjusted HR of Danshen users for >28 days was 0.55 [95% CI, 0.49–0.62] (p <0.001). Thus, these data demonstrate the protective effects of a higher dose or longer use of Danshen for patients with breast cancer. Question #4: Figure 2D, Figure 3H: (1). According to these results, curcumin increased ferrous ion levels in MCF-7 and MDA-MB-453 cells. However, curcumin has effective ferrous ions chelating capacity (Ak and Gulcin, Chemico-Biol Int 174, 27-37, 2008). In fact, curcumin reduces ferrous ions in cultured cells (Hirata et al., ACS Chem Neurosci 11, 76-85, 2020) and in in vitro assay (Shome et al., Biotechnology and Applied Biochemistry 68, 603-615 Biotechnology and Applied Biochemistry, 2021) at concentrations similar to those reported in this study. The authors need to examine the effect defferoxamine on ferrous ion levels as a positive control. Response #4(1): As suggested, we examined the effect deferoxamine (DFO) on the curcumin-induced increase in intracellular iron, and the results as showed in supplementary Fig. 2. We added this information in line 210-212, page 9 in this revision. (2). Please check whether the unit of ferrous ion is correct. Response #4(2): As suggested, we checked the unit of ferrous and revised this error in Figs 2 and 3 in this revision. (3). The relative of intracellular Fe2+ expression …: Intracellular Fe2+ level in both … Response #4(3): We corrected the “The relative of intracellular Fe2+ expression” into “The intracellular Fe2+ level”. (Line 483 and 493, page 17 in this revision) Minor points: Question #5: Figure 1: Is the concentration of erastin 10 nM? Response #5: Thanks for your carefully checking. In this study, the concentration of curcumin in both breast cancer cells was 10 nM. Question #6: Figure legends: The number of samples used in the experiments and statistics needs to be clarified. Response #6: We have added the detailed information for the samples used in the figure legend in this revision. (Line 477, 485, 494, 504 and 513, page 17-18 in this revision) Reviewer #2 In this article, the authors performed a series of in vitro and in vivo experiments to find out novel anti-tumorigenic mechanistic insight of curcumin. And they revealed that curcumin administration induced ferroptosis by enhancing the levels of lipid reactive oxygen species (ROS), malondialdehyde (MDA) of lipid peroxidation, and intracellular Fe2+ level in BC cells. Furthermore, treatment with curcumin significantly suppresses tumorigenesis in BC via upregulating SLC1A5 expression, which is an essential transporter for glutamine uptake. As a whole, the authors performed thorough experimental plan, and their finding are quite interesting. However, the authors need further improvement of this article to grown-up to publishing-quality. The followings are the issues that the authors should be addressed. Question #1: Because there are some other published articles in which Li R, et al, revealed the anti-tumorigenic effect of curcumin by altering other ferroptosis associated gene (HO-1) in breast cancer area (Oxid Med Cell Longev. 2020 Nov 18; 2020:3469840), the authors should add this article as reference and should add some discussion with regard to this article. Response #1: As suggested, we cited this reference as Ref.39 and added some discussion in line 304-308, page 12 in this revision. Question #2: As for Figure2A,2B, and 3F, please add the title of X-axis. Lipid ROS level? Response #2: As suggested, we added the title of X-axis as “Lipid ROS content” in Figure 2A, 2B and 3F. Question #3: How about changing the position of Figure 5F to 5A in order to explain the less-toxic effect of curcumin to mice? Response #3: As suggested, we exchanged the position of Figure 5F and 5A. Reviewer #3 In this article, the authors show that curcumin exhibits anti-tumorigenesis activity in breast cancer cell lines by promoting SLC1A5-mediated ferroptosis (lipid ROS, lipid peroxidation end-product MDA accumulation, and intracellular Fe2+ levels). Objectives are clear, experiments are well designed, and methodology is adequate to obtain the results. The article is well written. Major issues Question #1: Which diluent is used to dissolve curcumin? It must be checked that the vehicle does not reduce cells viability. Response #1: As suggested, we provided the dilution method of curcumin and test the cell viability of DMSO solvent. As shown in supplementary Fig. 1, the corresponding volume of DMSO had no cytotoxicity on MDA-MB-453 and MCF-7 cells (supplementary Fig. 1). (Line 86-88, page 4 and line 189-191, page 8 in this revision.) Question #2: Authors explain the role of ASCT2, SLC1A5 and SLC38A1 in glutamine uptake, but they only measured SLC1A5. Then they argue about the role of SLC1A5 in ferroptosis induced by curcumin but not about other receptors. Other receptors should be measured to confirm SLC1A5 implication. Response #2: ASCT2 is encoded by SLC1A5 that was evaluated in this original manuscript. As suggested, we further confirmed the mRNA expression of SLC38A1 in MDA-MB-453 and MCF-7 cells after treated with or without curcumin. As shown in supplementary Fig. 3, curcumin significantly enhanced the expression SLC1A5, but had no obvious effect on the expression of SLC38A1. We added this information in line 221-222, page 9 in this revision. Question #3: Immunohistochemistry staining should be quantified; a representative image is not enough to conclude Ki-67 and SLC1A5 increase. Response #3: As suggested, we provided the quantified results of Immunohistochemistry staining as Figure 5F in this revision. Question #4: To confirm the role of ferroptosis in vivo, authors should perform the same experiment but treating mice with ferrostatin and other cell death inhibitors, such as ZVAD and NS. In addition, authors should measure other ferroptosis markers than just MDA in the tumor to conclude the role of this pathway. Response #4: As suggested, to confirm that curcumin inhibited tumor growth in vivo by ferroptosis, we treated mice with curcumin, curcumin + DFO and curcumin + NS. As shown in Figure 5B-D in this revision, the administration of curcumin + DFO obviously reversed the inhibitory effect of curcumin on tumor growth, whereas the addition of NS, did not impact the efficiency of curcumin, which was consistent with the results of in vitro experiments that curcumin showed an antitumor effect on BC cells by inducing cell ferroptosis. (Line 248-255, page 10 in this revision.) In addition, we supplemented the results of other ferroptosis markers such as GSH and iron in tumor tissues of mice and the results were shown in Figure 5G-I. (Line 259-262, page 10 in this revision.) Question #5: In the discussion sections, authors report that curcumin promotes ferroptosis. However, there are contradictory data in the bibliography about this issue, see reference 38 (Guerrero-Hue et al.). Authors should discuss about the possible dual role of curcumin on ferroptosis. Response #5: Thanks for your comments, we have supplemented the possible dual role of curcumin on ferroptosis in the Discussion section. (Line 304-308, page 12 in this revision.) Question #6: Authors should add in the figure legend the time in which each experiment was performed. Response #6: As suggested, we added the treatment time in which each experiment in the figure legend. (Line 474, 476, 482 and 508, page 17-18 in this revision.) References in this letter: 1. Mohammed F, Rashid-Doubell F, Taha S, Cassidy S, Fredericks S. Effects of curcumin complexes on MDAMB231 breast cancer cell proliferation. Int J Oncol. 2020;57(2):445-55. Epub 2020/07/07. doi: 10.3892/ijo.2020.5065. PubMed PMID: 32626932; PubMed Central PMCID: PMCPMC7307592. 2. Hu CX, Li MJ, Guo TT, Wang SX, Huang WP, Yang K, et al. Anti-metastasis activity of curcumin against breast cancer via the inhibition of stem cell-like properties and EMT. Phytomedicine. 2019;58. doi: ARTN 152740 10.1016/j.phymed.2018.11.001. PubMed PMID: WOS:000473047100001. 3. Xiong K, Zhang Y, Wen Q, Luo J, Lu Y, Wu Z, et al. Co-delivery of paclitaxel and curcumin by biodegradable polymeric nanoparticles for breast cancer chemotherapy. Int J Pharm. 2020;589:119875. Epub 2020/09/13. doi: 10.1016/j.ijpharm.2020.119875. PubMed PMID: 32919003. 4. Kalluru H, Kondaveeti SS, Telapolu S, Kalachaveedu M. Turmeric supplementation improves the quality of life and hematological parameters in breast cancer patients on paclitaxel chemotherapy: A case series. Complement Ther Clin Pract. 2020;41:101247. Epub 2020/10/26. doi: 10.1016/j.ctcp.2020.101247. PubMed PMID: 33099272. 5. Wen C, Fu L, Huang J, Dai Y, Wang B, Xu G, et al. Curcumin reverses doxorubicin resistance via inhibition the efflux function of ABCB4 in doxorubicinresistant breast cancer cells. Mol Med Rep. 2019;19(6):5162-8. Epub 2019/05/07. doi: 10.3892/mmr.2019.10180. PubMed PMID: 31059026; PubMed Central PMCID: PMCPMC6522915. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0261370.r002
19 Oct 2021 Decision Letter - Irina V. Balalaeva, Editor PONE-D-21-20659R1Curcumin suppresses tumorigenesis by ferroptosis in breast cancerPLOS ONE Dear Dr. Guan, 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: Please, check the concentrations of the compounds in the experiments, especially those pointed at by the Reviewers. Please submit your revised manuscript by Dec 03 2021 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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Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. 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: (No Response) 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: No 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: The authors have not adequately addressed my concerns. Question #4/Response #4(1) (1) The authors did not discuss the chelating ability of curcumin and ignored the related references (Ak and Guicin, 2008; Hirata et al., 2020, Shome et al., 2021). The chelating ability of curcumin is opposite action the authors reported in this study and therefore the authors should discuss the discrepancy. The authors examined the effect of deferoxamine (DFO) on the curcumin-induced increase in intracellular Fe2+ (Figure S2). DFO only affected curcumin-induced increase in intracellular ferrous ions. The results is strange. Why DFO did not decrease the control level of ferrous ions? Question #4/Response #4(2) (2) The authors correct the unit of ferrous ion μg/μg protein to nmol/μg protein. According to the abcam’s protocol (ab83366, abcam) the authors used, rat liver lysate contains approximately 0.1 nmoles Fe2+/mg tissue, which is approximately 10,000 times less compared the amount of Fe2+ the authors reported (10 µmoles/µg protein). Question #5/Response #5 Erastin at 10 nM cannot cause ferroptotic cell death in cultured cells. Most cancer cells are required 5-10 µM erastin to cause ferroptosis. Reviewer #2: The authors have satisfactorily addressed the issues raised by reviewers and have improved the manuscript. Therefore the revised article has reached to sufficient quality. ****** 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 [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.0261370.r003
Revision 2
29 Oct 2021 Author Response Response to reviewer Reviewer #1: Question #4/Response #4(1): The authors did not discuss the chelating ability of curcumin and ignored the related references (Ak and Guicin, 2008; Hirata et al., 2020, Shome et al., 2021). The chelating ability of curcumin is opposite action the authors reported in this study and therefore the authors should discuss the discrepancy. The authors examined the effect of deferoxamine (DFO) on the curcumin-induced increase in intracellular Fe2+ (Figure S2). DFO only affected curcumin-induced increase in intracellular ferrous ions. The results are strange. Why DFO did not decrease the control level of ferrous ions? Response: We felt great thanks for your professional review work on our article. As suggested, we added the related references (Ak and Guicin, 2008; Hirata et al., 2020, Shome et al., 2021) as Ref. 41-43 and discussed the discrepancy between these studies and our work (line 308-318, page 12 in this revision). In addition, as shown in supplementary Fig. 2, our data showed that DFO did not only affect curcumin-induced increase in intracellular ferrous ions, but also decrease the control level of ferrous ions (the second column). Question #4/Response #4(2): The authors correct the unit of ferrous ion μg/μg protein to nmol/μg protein. According to the abcam’s protocol (ab83366, abcam) the authors used, rat liver lysate contains approximately 0.1 nmoles Fe2+/mg tissue, which is approximately 10,000 times less compared the amount of Fe2+ the authors reported (10 µmoles/µg protein). Response: In this study, the unit of ferrous was (nmol/μg protein) × 103 in Fig. 2 and 3, which is equivalent to nmol/mg protein. Question #5/Response #5: Erastin at 10 nM cannot cause ferroptotic cell death in cultured cells. Most cancer cells are required 5-10 µM erastin to cause ferroptosis. Response: We are very sorry for our carelessness in writing. After careful checking, we verified that the dose of erastin used in this experiment was 10 µM. Special thank you very much for your careful review and we express our sincere apologies for this error. We corrected this mistake in this revision (line 90, page 4 and line 501, page 18 in this revision). Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0261370.r004
12 Nov 2021 Decision Letter - Irina V. Balalaeva, Editor PONE-D-21-20659R2Curcumin suppresses tumorigenesis by ferroptosis in breast cancerPLOS ONE Dear Dr. Guan, 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: Please, check the results mentioned by the Reviewer. Please submit your revised manuscript by Dec 27 2021 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, Irina V. Balalaeva, PhD 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 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 ******** 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The author’s response: In addition, as shown in supplementary Fig. 2, our data showed that DFO did not only affect curcumin-induced increase in intracellular ferrous ions, but also decrease the control level of ferrous ions (the second column). The authors did not respond my concern as follows: DFO only affected curcumin-induced increase in intracellular ferrous ions (Figure S2A, the 3rd column vs 4th column) but did not affect the control level of ferrous ions (Figure S2A, the first column vs second column). The results is strange. The English writing standard needs to be improved further. ****** 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 [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.0261370.r005
Revision 3
24 Nov 2021 Author Response Reviewer #1: Question #1: The authors did not respond my concern as follows: DFO only affected curcumin-induced increase in intracellular ferrous ions (Figure S2A, the 3rd column vs 4th column) but did not affect the control level of ferrous ions (Figure S2A, the first column vs second column). The results are strange. Response #1: We retest the intracellular ferrous ions in MDA-MB-453 and MCF-7 cells with or without the presence of DFO. As shown in Figure 1 in this response, no obvious difference was observed between the intracellular ion level in of MDA-MB-453 and MCF-7 cells with or without the presence of 50 μM deferoxamine, which was also found in recent published researches [1-3]. Although deferoxamine mesylate was an iron chelator that bound free iron in a stable complex, we speculated that it might not affect the relatively low normal ferrous ions level in some cells but effectively reverse the iron accumulation induced by ferroptosis inducer. Question #2: The English writing standard needs to be improved further. Response #2: As suggested, we improved the quality of our language by an English language editing service of Spandidos Publications. Based on the editing report, we revised our manuscript and also uploaded the certificate with this revision. References in this response: 1. Tu H, Zhou YJ, Tang LJ, Xiong XM, Zhang XJ, Ali Sheikh MS, et al. Combination of ponatinib with deferoxamine synergistically mitigates ischemic heart injury via simultaneous prevention of necroptosis and ferroptosis. Eur J Pharmacol. 2021;898:173999. Epub 2021/03/07. doi: 10.1016/j.ejphar.2021.173999. PubMed PMID: 33675785. 2. Yang J, Zhou Y, Xie S, Wang J, Li Z, Chen L, et al. Metformin induces Ferroptosis by inhibiting UFMylation of SLC7A11 in breast cancer. J Exp Clin Cancer Res. 2021;40(1):206. Epub 2021/06/25. doi: 10.1186/s13046-021-02012-7. PubMed PMID: 34162423; PubMed Central PMCID: PMCPMC8223374. 3. Song Z, Xiang X, Li J, Deng J, Fang Z, Zhang L, et al. Ruscogenin induces ferroptosis in pancreatic cancer cells. Oncol Rep. 2020;43(2):516-24. Epub 2020/01/03. doi: 10.3892/or.2019.7425. PubMed PMID: 31894321; PubMed Central PMCID: PMCPMC6967081. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0261370.r006
1 Dec 2021 Decision Letter - Irina V. Balalaeva, Editor Curcumin suppresses tumorigenesis by ferroptosis in breast cancer PONE-D-21-20659R3 Dear Dr. Guan, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Irina V. Balalaeva, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0261370.r007
Formally Accepted
7 Jan 2022 Acceptance Letter - Irina V. Balalaeva, Editor PONE-D-21-20659R3 Curcumin suppresses tumorigenesis by ferroptosis in breast cancer Dear Dr. Guan: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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 plosone@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 Dr. Irina V. Balalaeva Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0261370.r008

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