3,3’-Diindolylmethane induces apoptosis and autophagy in fission yeast

Parvaneh Emami; Masaru Ueno

doi:10.1371/journal.pone.0255758

Peer Review History

Original SubmissionJuly 20, 2021
24 Aug 2021 Decision Letter - Reiko Sugiura, Editor PONE-D-21-23534 3,3'-Diindolylmethane induces apoptosis and autophagy in fission yeast PLOS ONE Dear Dr. Ueno, 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. Both reviewers felt that the area of investigation was of interest and recognize that there is a substantial body of work represented in the manuscript. However, they had some concerns about the depth or rigorousness of the analysis of the study in its current form. These include the quality of the dilution tests, validity of the drug effects for long-time incubation, as well as the lack of appropriate control strains in each experiment, statistical analysis, or the logic of the experiments. 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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: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: No ****** 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 this manuscript, the authors examine the effect of 3,3'-diindolylmethane, which has potential anti-cancer properties, in the fission yeast S. pombe. High concentrations of DIM induce apoptosis-like cell death in log-phase cells, while stationary-phase cells are resistant to high dosage of DIM. DIM impairs nuclear envelope integrity, and a knock-out mutant of nuclear membrane protein Lem2 shows higher sensitivity to DIM than wild-type cells. DIM also induces autophagy at a low concentration, as shown in human cells. Overall, the experiments are well conducted and the data presented is informative. Specific points: 1. Figure 1A shows that DIM inhibits fission yeast growth only for 1 day. The authors speculate from this data that "DIM does not inhibit growth when cells are in the stationary-phase" (Lines 203-204). It is difficult to draw the conclusion from Figure 1A, while this speculation seems correct from the other data such as Figure 1B and C. Do surviving stationary-phase cells grow in the presence of DIM? It is likely that DIM toxicity is lost after day 2 in the experiment of Figure 1A. The growth curve for the first 24 hours in the high concentration of DIM may be informative. 2. Figure 2C. Frequencies of cells with fragmented nucleus should be shown. 3. Lines 308-309 read that 10-minute DIM treatment can induce apoptosis. Does 10-minute incubation induce nuclear fragmentation? Reviewer #2: SUMMARY: DIM induces apoptosis and autophagy in a fission yeast. The authors observe that a high concentration of DIM disrupted the nuclear envelope and induced chromosomal condensation. This did not happen with a low concentration of DIM. Instead the cells were triggered to undergo autophagy. A atg7 mutant defective in autophagy was more sensitive to low doses of DIM. Similarly, a lem2 mutant defective in NE physiology was also more sensitive to the drug. Comments on the Figures: Figure 1A: Providing two dilution spots is not typical. The authors should provide at least four dilution spots so that they can quantify the loss of viability. Provide statistical analysis. When the authors comment that DIM did not inhibit growth after additional incubation, suggesting that DIM does not inhibit growth when cells are in stationary phase, how do they rule out the alternative explanation that DIM simply degrades within 24 hours and such is not present to mitigate cell growth after 1 day. Do they know the half-life of DIM in a DMSO solution? How do the authors know that the DIM is still present at day 5 or day 9? 1. Figure 1B: Once again, the authors need to provide at least four dilution spots so that they can quantify the viability of the cells and graph it. Provide statistical analysis. Also, this panel is not easy to understand. More labels are needed. Figure 1C: Once again, the authors need to provide at least four dilution spots so that they can quantify the viability of the cells and graph it. Provide statistical analysis. Figure 2: Can the authors include a positive control that is known to induce apoptosis in fission cells? Otherwise it is hard to know how to interpret the figures. Figure 3: Can the authors include a positive control that is known to induce nuclear condensation and NE disruption in fission cells? Otherwise it is hard to know how to interpret the figures. Can the authors quantify the NE disruption in Figures 3C and 3D in the same way they quantified 3A in 3B? Provide statistical analysis. Figure 5A: Can the authors include a positive control that is known to induce autophagy in fission cells to show lethality of the atg7 mutant? Also, can they quantify the loss of viability as they did in Figure 2B, for example. Provide statistical analysis. Figure 5B: Why did the authors expect autophagy induction by DIM to be dependent on Ire1p? The authors need to describe their expectations that link ER-stress and autophagy in the manuscript. Figure 6A: Can the authors quantify the loss of viability and graph it? Provide statistical analysis. Figure 6B: Can the authors quantify loss of nuclear envelope integrity and graph it? Provide statistical analysis. ****** 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 [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. 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Revision 1
15 Nov 2021 Author Response Response to Reviewer1 Reviewer #1: In this manuscript, the authors examine the effect of 3,3'-diindolylmethane, which has potential anti-cancer properties, in the fission yeast S. pombe. High concentrations of DIM induce apoptosis-like cell death in log-phase cells, while stationary-phase cells are resistant to high dosage of DIM. DIM impairs nuclear envelope integrity, and a knock-out mutant of nuclear membrane protein Lem2 shows higher sensitivity to DIM than wild-type cells. DIM also induces autophagy at a low concentration, as shown in human cells. Overall, the experiments are well conducted and the data presented is informative. (1) Response to [1. Figure 1A shows that DIM inhibits fission yeast growth only for 1 day. The authors speculate from this data that "DIM does not inhibit growth when cells are in the stationary-phase" (Lines 203-204). It is difficult to draw the conclusion from Figure 1A, while this speculation seems correct from the other data such as Figure 1B and C. Do surviving stationary-phase cells grow in the presence of DIM? It is likely that DIM toxicity is lost after day 2 in the experiment of Figure 1A. The growth curve for the first 24 hours in the high concentration of DIM may be informative.] (1-1) Response to: ["DIM does not inhibit growth when cells are in the stationary-phase" (Lines 203-204). It is difficult to draw the conclusion from Figure 1A.] I agree to the reviewer’s comment. Therefore, we revised sentence as shown below. Previous: DIM did not inhibit growth after additional incubation, suggesting that DIM does not inhibit growth when cells are in the stationary-phase. Page 15 lines 226-231 Revised: DIM did not inhibit growth after additional incubation, suggesting that either DIM does not decrease the viability when cells are in the stationary-phase or DIM is decomposed. To test the possibility that DIM does not decrease the viability when cells are in the stationary-phase, we added DIM to the cells that were in stationary-phase. (1-2) Response to: [Do surviving stationary-phase cells grow in the presence of DIM? It is likely that DIM toxicity is lost after day 2 in the experiment of Figure 1A.] As suggested by reviewer, we asked whether the surviving stationary-phase cells grow in the presence of DIM. We found that the surviving stationary-phase cells cannot grow in the presence of DIM. This result suggests that either DIM is decomposed in 2 days or surviving stationary-phase cells tried to return to log-phase cell on DIM plate and became sensitive to DIM. Stephan et. al. Aging Cell (2013) shows that pka1 mutant is sensitive to DIM even after 3 days incubation with DIM, suggesting that DIM is still active after 3 days incubation. However, it is still possible that DIM could be partially decomposed after 2 days. Our main question is acute effect of DIM. Therefore, we will focus on acute effect of DIM. We have a hypothesis that log phase cells become resistant to DIM when cells are incubated in the presence of DIM after 24 h possibly by epigenetic adaptation. These DIM resistant cells can reach to stationary phase. This epigenetic adaptation is erased when the surviving stationary-phase cells are incubated in fresh YEA plate with DIM. This hypothesis will be studied in another manuscript. Therefore, we do not want to include data showing that the surviving stationary-phase cells can not grow in the presence of DIM in our current manuscript. (1-3) Response to: [The growth curve for the first 24 hours in the high concentration of DIM may be informative.] As suggested reviewer, we added the growth curve for the first 24 hours in the high concentration of DIM in SFig. S1 and text in page 15 line 226). Cell number did not increase first 24 hours in the high concentration of DIM. (2) Response to: [2. Figure 2C. Frequencies of cells with fragmented nucleus should be shown.] As suggested by reviewer, we added the frequencies of cells with fragmented nucleus in new Fig. 3D. (3) Response to: [3. Lines 308-309 read that 10-minute DIM treatment can induce apoptosis. Does 10-minute incubation induce nuclear fragmentation?] As suggested by reviewer, we testes this point and found that at 10 min incubation with DIM induce nuclear fragmentation after 6h. We added the frequencies of cells with fragmented nucleus in the new Fig. S4B to mention that 10 min incubation with DIM induce nuclear fragmentation after 6h. To explain this, we added sentence below. Added sentence page 26 lines 425-429 However, this 10-minute treatment resulted in nuclear fragmentation after 6 hours (S4B Fig) and loss the viability (Figs. 2A and B), suggesting that 10 minutes of incubation with DIM is enough to induce apoptosis and the process of apoptotic cell death continues even after DIM removal. (4) Additional modification that was not suggested by reviewer #1. We removed previous Fig. 1B log phase data, because it was a repetition of previous Fig. 1A. We also removed previous Fig. 6B 10 min data, because there was no change at all. Moreover, these data were the repetition of previous Fig. 1C and D. We also removed previous Fig. S1B data, because it was a repetition of previous Fig. 1C. Response to Reviewer2 Reviewer #2: SUMMARY: DIM induces apoptosis and autophagy in a fission yeast. The authors observe that a high concentration of DIM disrupted the nuclear envelope and induced chromosomal condensation. This did not happen with a low concentration of DIM. Instead the cells were triggered to undergo autophagy. A atg7 mutant defective in autophagy was more sensitive to low doses of DIM. Similarly, a lem2 mutant defective in NE physiology was also more sensitive to the drug. Comments on the Figures: (1) Response to: [Figure 1A: Providing two dilution spots is not typical. The authors should provide at least four dilution spots so that they can quantify the loss of viability. Provide statistical analysis. When the authors comment that DIM did not inhibit growth after additional incubation, suggesting that DIM does not inhibit growth when cells are in stationary phase, how do they rule out the alternative explanation that DIM simply degrades within 24 hours and such is not present to mitigate cell growth after 1 day. Do they know the half-life of DIM in a DMSO solution? How do the authors know that the DIM is still present at day 5 or day 9?] (1-1) Response to [The authors should provide at least four dilution spots so that they can quantify the loss of viability.] As suggested by reviewer, we provided four dilution spots in all data (Fig. 1A and C, Fig. 2A, Fig. 6A and B, Fig. 7A). (1-2) Response to [Provide statistical analysis] As suggested by reviewer, we quantitated dada and provided statistical analysis for all data (Fig. 1B and D, Fig. 2B and C, Fig. 3B and D, Fig. 4D and E, Fig. 6C, Fig. 7B and D). (1-3) Response to: [When the authors comment that DIM did not inhibit growth after additional incubation, suggesting that DIM does not inhibit growth when cells are in stationary phase, how do they rule out the alternative explanation that DIM simply degrades within 24 hours and such is not present to mitigate cell growth after 1 day. Do they know the half-life of DIM in a DMSO solution? How do the authors know that the DIM is still present at day 5 or day 9?] I agree to reviewer’s comment. Stephan et. al. Aging Cell (2013) shows that pka1 mutant is sensitive to DIM even after 3 days incubation with DIM, suggesting that DIM is still active after 3 days incubation. However, it is still possible that DIM could be partially decomposed after two days. Our main question is acute effect of DIM. Therefore, we will focus on acute effect of DIM. We revised sentence as shown below. Previous: DIM did not inhibit growth after additional incubation, suggesting that DIM does not inhibit growth when cells are in the stationary-phase. Page 15 lines 226-231 Revised: DIM did not inhibit growth after additional incubation, suggesting that either DIM does not decrease the viability when cells are in the stationary-phase or DIM is decomposed. To test the possibility that DIM does not decrease the viability when cells are in the stationary-phase, we added DIM to the cells that were in stationary-phase. (2-1) Response to: [Figure 1B: Once again, the authors need to provide at least four dilution spots so that they can quantify the viability of the cells and graph it. Provide statistical analysis. Also, this panel is not easy to understand.] Same response as shown above in (1-1) and (1-2). (2-2) Response to: [More labels are needed.] As suggested by reviewer, we provided labels to show the difference between log phase and stationary phase cells in Fig. 1 and 2. To make Figure 1 more easily understandable, we separated data to new Fig.1 (chronic assay) and new Fig. 2 (acute assay). Moreover, log-phase data in previous Fig. 1B had repetition to previous Fig. 1A. Therefore, we removed log-phase data that was repeatedly shown in previous Fig. 1B. We also changed the position of spot assay picture from up-down to side-by-side with new labels (log-phase cells and stationary phase cells) in new Fig. 2A. (3) Response to: [Figure 1C: Once again, the authors need to provide at least four dilution spots so that they can quantify the viability of the cells and graph it. Provide statistical analysis.] Same response as shown above in (1-1) and (1-2). (4) Response to: [Figure 2: Can the authors include a positive control that is known to induce apoptosis in fission cells? Otherwise it is hard to know how to interpret the figures.] As suggested by reviewer, we included a positive control called Terpinolene that is known to induce apoptosis and shown in new Fig. 3 and text page 18 line 278 and 285). (5)　Response to:　[Figure 3: Can the authors include a positive control that is known to induce nuclear condensation and NE disruption in fission cells? Otherwise it is hard to know how to interpret the figures.] It is impossible to provide positive control that induce nuclear condensation and NE disruption within 10 minutes because there is no paper showing that nuclear condensation and NE disruption are induced 10 min. after drug addition. To our knowledge, this is completely new experiment and new data. Therefore, we added sentences shown below. Added sentence to page 26 lines 432-433 To our knowledge, these phenotypes are not shown in fission yeast so far. (6) Response to: [Can the authors quantify the NE disruption in Figures 3C and 3D in the same way they quantified 3A in 3B? Provide statistical analysis.] Same response as shown above in (1-2). (7) Response to: [Figure 5A: Can the authors include a positive control that is known to induce autophagy in fission cells to show lethality of the atg7 mutant?] Yes, we added data showing that atg7 mutant is more sensitive to nitrogen starvation in SFig. 2 and text page 21 line 345, because it is already published (Fukuda et al. 2020), we showed this data in SFig. 2. (8) Response to: [Also, can they quantify the loss of viability as they did in Figure 2B, for example. Provide statistical analysis.] Same response as shown above in (1-2). (9) Response to: [Figure 5B: Why did the authors expect autophagy induction by DIM to be dependent on Ire1p? The authors need to describe their expectations that link ER-stress and autophagy in the manuscript.] It is reported that ER stress response is required for autophagy induction in ovarian cancer cells. Therefore, we investigated if ER stress response is required for autophagy induction by DIM in fission yeast. To explain it, we added following sentence. Added sentence in page 23 lines 374-378 However, ER stress inhibitor, mithramycin, blocks DIM mediated ER stress and subsequently inhibits autophagy induction, suggesting that ER stress response is required for autophagy induction in ovarian cancer cells [6]. Therefore, we investigated if ER stress response is required for autophagy induction by DIM in fission yeast. (10) Response to: [Figure 6A: Can the authors quantify the loss of viability and graph it? Provide statistical analysis.] Same response as shown above in (1-2). (11) Response to: [Figure 6B: Can the authors quantify loss of nuclear envelope integrity and graph it? Provide statistical analysis.] Same response as shown above in and (1-2). (12) Additional modification that was not suggested by reviewer 2. We removed previous Fig. 6B 10 min data, because there is no change at all. Moreover, these data were the repetition of previous Fig. 1C and D. We also removed previous Fig. S1B data, because it was a repetition of previous Fig. 1C. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0255758.r002
25 Nov 2021 Decision Letter - Reiko Sugiura, Editor 3,3'-Diindolylmethane induces apoptosis and autophagy in fission yeast PONE-D-21-23534R1 Dear Dr. Ueno, 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, Reiko Sugiura, M.D., PhD. Academic Editor PLOS ONE Additional Editor Comments (optional): 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: 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: (No Response) ****** 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 https://doi.org/10.1371/journal.pone.0255758.r003
Formally Accepted
2 Dec 2021 Acceptance Letter - Reiko Sugiura, Editor PONE-D-21-23534R1 3,3'-Diindolylmethane induces apoptosis and autophagy in fission yeast Dear Dr. Ueno: 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. Reiko Sugiura Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0255758.r004

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