Review Reports - Sirtuin Upregulation in the Cochlea During Honokiol-Mediated Protection Against Cisplatin Ototoxicity

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript addresses an important and clinically relevant question: whether honokiol can protect against cisplatin-induced ototoxicity without compromising its antitumor activity. The use of a tumor-bearing mouse model, longitudinal ABR measurements, and assessment of cochlear ribbon synapses are important strengths. In particular, the finding that honokiol preserves IHC ribbon synapses following cisplatin treatment is interesting and potentially represents one of the most valuable findings of the study. However, several aspects of the experimental design and interpretation require clarification. In particular, the mechanistic conclusions regarding sirtuin activation appear stronger than warranted by the presented data. I recommend major revision.

Major comments

The manuscript attributes the protective effects of honokiol largely to sirtuin activation. However, the presented data mainly demonstrate changes in sirtuin expression/immunoreactivity rather than enzymatic activity. Increased immunofluorescence signal or mRNA expression does not necessarily indicate increased sirtuin activity, nor does it establish a causal relationship between sirtuins and otoprotection. The authors should either provide additional functional evidence supporting a causal role of sirtuins or substantially moderate the terminology and conclusions throughout the manuscript. In particular, “increased expression” or “increased immunoreactivity” may be more appropriate than “activation” unless direct evidence of enzymatic activation is available. The authors should also discuss alternative mechanisms through which honokiol may protect the cochlea, including its potential effects on oxidative stress, mitochondrial dysfunction, inflammation, apoptosis, and other cellular stress-response pathways. Given the pleiotropic activity of honokiol, sirtuin-related changes may represent only one component of its protective effects.

Honokiol was administered 1 h before cisplatin. The rationale for selecting this specific time interval should be explained. Was it based on pharmacokinetic data, tissue distribution, previous studies, or the expected time required to activate protective pathways? The rationale for selecting 5–20 mg/kg honokiol should also be provided. The authors should discuss the translational relevance of these doses, including their relationship to previously reported pharmacologically active doses and, where appropriate, human-equivalent doses. Direct comparison with doses used in dietary supplements should be interpreted cautiously because of differences in administration route, bioavailability, and pharmacokinetics.

The manuscript mentions health issues occurring during the experiment, but these are not described in sufficient detail. Please specify the nature, timing, severity, and frequency of these events and indicate their distribution among treatment groups. The reasons for animal exclusion or euthanasia should be reported individually or at least by treatment group, with clarification as to whether the events were related to tumor progression, cisplatin, honokiol, or the combination treatment. This is particularly important because substantial animal attrition may influence the interpretation of the final ABR and histological analyses. Please also clarify how frequently body weight and general condition were monitored. Given the repeated cisplatin treatment and observed health problems, systematic monitoring of body weight would be important for evaluating treatment-related toxicity and animal welfare.

The statistical analysis of the repeated ABR measurements requires greater clarification. Please provide the exact specification of the linear mixed-effects model, including fixed and random effects, interaction terms, treatment of missing observations, and the contrasts used for statistical comparisons. The number of animals analyzed at each time point and frequency should also be clearly reported. Since not all animals completed the study, the potential impact of attrition/survivor bias on the results should be discussed.

The statement that honokiol is “synergistic” with cisplatin in tumor suppression is not sufficiently supported by the current analysis. A greater effect of the combination compared with cisplatin alone does not by itself demonstrate pharmacological synergy. Formal analysis of drug interaction would be required. Given the relatively small sample sizes in some treatment groups, the authors should either provide an appropriate synergy/interaction analysis or replace “synergistic” with more cautious terminology describing an enhanced or additional antitumor effect.

The observation that honokiol prevents cisplatin-induced loss of IHC ribbon synapses is particularly interesting and should be emphasized as an important finding of the study. At the same time, the authors should discuss more explicitly how this effect could be mechanistically related to the proposed sirtuin pathway and whether it may also result from sirtuin-independent effects of honokiol.

Minor comments

The abbreviations and treatment group designations, such as HNK5+Cis, HNK10+Cis, HNK20+Cis, and MMTV-PyMT, are explained in Table 1; however, they should also be defined at their first appearance in the main text to facilitate readability and avoid requiring the reader to refer to the table.
Please check the consistency of the honokiol doses and group names throughout the manuscript. In particular, the HNK20-Only group in Table 1 appears to be described as receiving 10 mg/kg/day, despite the group designation suggesting a dose of 20 mg/kg. Please clarify and correct this discrepancy, if applicable.
The terminology “nucleic sirtuin” should be checked; “nuclear sirtuin” may be intended.
The interpretation of the Cytc results should be more cautious. Changes in Cytc mRNA expression alone do not directly demonstrate reduced cytochrome c release or inhibition of mitochondrial apoptosis.
Please clarify whether quantitative immunofluorescence analyses were performed across biological replicates and provide the corresponding sample sizes and statistical analyses.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript deals with a knowledge gap related to the hearing loss side effect of cisplatin which is often overlooked compared to other side effects. The study is timely, comprehensive, and reports the protective effect of a natural compound "honokiol", which is novel. References are adequate in number and relevant to the study. Only few comments to be addressed before the manuscript can be accepted for publication:

1-Introduction: In one paragraph kindly summarize what has been reported in the literature till current date related to honokiol/cisplatin joint use, as this will better highlight the novelty of the study.

2- Materials: Please report the purity of honokiol, and the source of corn oil.

3-Please report the starting weight of the utilized mice.

4- Figure 3B shows high variability and substantial animal loss, particularly in cisplatin only group. Please report individual weight changes and the number of animals at each time point, and address whether removing significantly affected animals introduced survival bias or not.

5- Figures 6-8: the authors based the claim of increased sirtuin expression on qualitative and quantitative fluoresence analysis, so please acknowledge that this is a limitation of the current study.

Author Response

Reviewer 2

Thanks for the suggestion. To maintain conciseness and logic flow in the Introduction, we have expanded the third paragraph to include further context on honokiol to highlight the novelty of using honokiol for treating CIHL. We then expend on the joint use of honokiol and cisplatin and put updated information related to honokiol in the Discussion.

In the introduction, we added: “We previously showed that Sirt3 was activated during hearing protection against cisplatin ototoxicity using honokiol, a multifunctional natural compound and dietary supplement. As a reported Sirt3 activator, honokiol shows protective effects against oxidative damage in multiple tissues and enhanced tumor suppression effect that is compatible with cisplatin. These studies suggest that honokiol is a promising candidate for treating CIHL, and Sirt3 might be involved in the mechanism of this honokiol-mediated protection.”

In the Discussion, we added: “More importantly, honokiol can enhance conventional anticancer agents, and therefore is potentially a viable adjunct in combination therapies with cisplatin [40]. It has been shown that honokiol is synergistic with cisplatin in multiple cancer models, including colon cancer [65], ovarian cancer [66], and oral squamous cell carcinoma [67]. Me-chanically, Sirt3 activation seems to play critical roles [39,68,69], and honokiol can also sensitize cisplatin-resistant phenotype through modulating IL-6/Stat3 signaling [67], a critical pathway involved in tumorigenesis and development [70]. It is also shown that honokiol mitigates severe systemic toxicities, such as acute kidney injury, induced by cisplatin [71-73]. A recent review has summarized the application of honokiol in com-bination therapy for cancer treatment [40].”

In addition, we also changed slightly in the Abstract to highlight the synergistic effect of honokiol with cisplatin: “We previously reported that honokiol, a multifunctional molecule synergistic with cisplatin, protects against CIHL and upregulates sirtuin 3.”

2- Materials: Please report the purity of honokiol, and the source of corn oil.

Thanks for the suggestion. The information is provided in the revision.

3-Please report the starting weight of the utilized mice.

Sorry for the confusion. We understand the importance of reporting the original weight, and this was reported in Supplementary Materials (Figure S3). We kept it in the same place in the revision to maintain the flow of the main text.

Again, the weight changes were reported in Supplementary Materials, Figure S3. The numbers of animals at each time points (before the treatment and at the end of each cycle) are indicated by the Survival Rate (%) in Figure 3A and the number of animals in each cycle in Figure S1 and S2. We summarized the survival rates in the Supplementary data with an extra Table S1.

We greatly appreciate the potential survival bias issue raised by the reviewer. We discussed the potential influence of the criteria for weight loss on the hearing threshold and acknowledged that this could be one of the limitations of the study. It is possible that the mice most susceptible to the systemic toxicity of cisplatin were also those that would have exhibited the most severe hearing loss. We have now explicitly stated this potential survival bias in the Discussion. We added: “Because these animals did not survive to the end of the third cycle, our reported ABR thresholds may have introduced a survival bias which underestimated the true severity of cisplatin ototoxicity.”

5- Figures 6-8: the authors based the claim of increased sirtuin expression on qualitative and quantitative fluoresence analysis, so please acknowledge that this is a limitation of the current study.

Thanks for the suggestion. We are aware that immunofluorescence intensity can be affected by various factors, and therefore the analysis should only be considered quasi-quantitative for the abundance of proteins. This is also one of the reasons why we conducted RT-qPCR to confirm on RNA level the findings on sirtuin expression. We added the following statement in the limitation of the study:

“Third, immunofluorescence intensity can be affected by various factors such as antibody affinity, tissue penetration, and photobleaching. Therefore, sirtuin expression and upregulation evaluated with fluorescence intensity should only be considered quasi-quantitative and interpreted as relative changes rather than quantitative changes in protein concentration. Upregulation in sirtuin protein levels does not necessarily indicate increased enzymatic activity, either.”

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript entitled “Sirtuin Activation in the Cochlea during Honokiol-Mediated Protection Against Cisplatin Ototoxicity” addresses an interesting and clinically relevant topic. The study is generally well designed, and the methodology is appropriate for the stated objectives.

Overall, the manuscript is well written and scientifically sound. However, several issues should be addressed before it can be considered for publication:

The Introduction should provide additional background information on honokiol. The authors should briefly describe this naturally occurring chemical compound, including its biological and pharmacological activities relevant to the present study.
The term “sirtuin activation,” which is used in the title and throughout the manuscript, may be too strong considering the presented evidence. The study mainly demonstrates changes in immunofluorescence signals and mRNA expression, whereas direct sirtuin enzymatic activity was not measured.
In Section 4.1, Ethics Statement, the country should be added after “Northwestern University.”
The source of the experimental mice should be clearly stated, including the supplier or breeding facility and its location.
The reported improvement in animal survival and weight change (Figure 3) should be supported by an appropriate statistical comparison.
The authors describe the interaction between honokiol and cisplatin as “synergistic” based on tumor growth suppression. However, greater tumor suppression following combined treatment alone does not necessarily demonstrate pharmacological synergy. A suitable interaction analysis should be provided, or the term “synergistic” should be replaced with a more cautious expression such as “enhanced” or “additional antitumor effect.”
The numbering of the Results subsections should be corrected, as Section 2.2.2 is followed directly by Section 2.4.
In the legend of Figure 9, **p < 0.01 is indicated; however, it is not specified which groups were compared. Please clearly state the corresponding comparison in the figure legend.
The subheadings in the Discussion section are not necessary and may be removed to improve the continuity and readability of the section.
A separate Conclusion section should be added. It should concisely summarize the principal findings and their significance while avoiding causal or mechanistic claims that are not directly supported by the presented results.

Author Response

Reviewer 3

Overall, the manuscript is well written and scientifically sound. However, several issues should be addressed before it can be considered for publication:

The Introduction should provide additional background information on honokiol. The authors should briefly describe this naturally occurring chemical compound, including its biological and pharmacological activities relevant to the present study.

We greatly appreciate this suggestion, which was raised by both Reviewers 2 and 3. As we addressed in Comment 1 of Reviewer 2, we made relevant changes in Abstract, Introduction, and Discussion.

The term “sirtuin activation,” which is used in the title and throughout the manuscript, may be too strong considering the presented evidence. The study mainly demonstrates changes in immunofluorescence signals and mRNA expression, whereas direct sirtuin enzymatic activity was not measured.

Again, we greatly appreciate this suggestion as well and changed the word “activation” into “upregulation” wherever appropriate. In addition, we specifically added a statement in the last part of the Discussion (limitation of the study) that: “Upregulation in sirtuin protein levels does not necessarily indicate increased enzymatic activity, either.”

In Section 4.1, Ethics Statement, the country should be added after “Northwestern University.”

The country name USA is added wherever appropriate. Specifically, it is added in 4.1. Ethics statement, Institutional Review Board Statement, and Conflicts of Interest.

The source of the experimental mice should be clearly stated, including the supplier or breeding facility and its location.

Sorry for the ignorance. The information of mouse strains is now included in 4.2. Animal groups and treatments.

The reported improvement in animal survival and weight change (Figure 3) should be supported by an appropriate statistical comparison.

Thanks for the comments. For the weight change, we have rerun the statistical analysis using Welch’s ANOVA to replace the conventional one-way ANOVA due to relatively small and unequal group sizes. For survival rate, we specifically stated that “Kaplan-Meier survival analysis was not performed due to incomplete recording of daily animal loss.” In the revision, we conducted a 4-group contingency analysis on cycle 3. We added in the text:

“Since the group sizes were relatively small and unequal, a more conservative Welch’s ANOVA was performed (F(3,12.43) = 4.05, P = 0.0323). A following Games–Howell post-hoc testing showed that the weight losses were significantly lower in honokiol co-treated groups (p = 0.024, 0.254, and 0.028 for honokiol 5, 10, and 20 mg/kg, respectively, compared to Cis-Only group) (Fig. 3B). The values of animal weight during the treatment were reported in Fig. S3. No significant difference in pre-treatment baseline weight was detected in different treatment groups.

Kaplan–Meier survival analysis was not performed because the exact timing of individual animal losses was not recorded consistently throughout the study. Instead, survival of the four cisplatin-treated groups at the end of Cycle 3 was compared using a 4-group contingency analysis. Although the proportion of surviving animals was numerically higher in the honokiol co-treatment groups than in the Cis-Only group, the overall difference among groups was not statistically significant (χ²(3) = 1.36, P = 0.715).”

The authors describe the interaction between honokiol and cisplatin as “synergistic” based on tumor growth suppression. However, greater tumor suppression following combined treatment alone does not necessarily demonstrate pharmacological synergy. A suitable interaction analysis should be provided, or the term “synergistic” should be replaced with a more cautious expression such as “enhanced” or “additional antitumor effect.”

Thanks for the comment. We replaced the word “synergistic” to “compatible”, “enhancement”, and “additional antitumor effect” where appropriate.

The numbering of the Results subsections should be corrected, as Section 2.2.2 is followed directly by Section 2.4.

Sorry for the ignorance. It is now corrected.

In the legend of Figure 9, **p < 0.01 is indicated; however, it is not specified which groups were compared. Please clearly state the corresponding comparison in the figure legend.

Relative expression was calculated using the 2^(-ΔΔC_t ) method and Actb as reference. In figure 9, pairwise comparisons were between the honokiol-treated group versus untreated vehicle controls. We thank the reviewer for identifying this problem and we have updated the legend for Figure 9.

The subheadings in the Discussion section are not necessary and may be removed to improve the continuity and readability of the section.

It is now corrected.

A separate Conclusion section should be added. It should concisely summarize the principal findings and their significance while avoiding causal or mechanistic claims that are not directly supported by the presented results.

Conclusions are added as suggested.

“This study demonstrated that honokiol mitigates CIHL in a tumor-bearing mouse model without compromising therapeutic efficacy of cisplatin. Pretreatment with honokiol significantly attenuated ABR threshold shifts, loss of ribbon synapse, and health deterioration induced by cisplatin treatment. This honokiol-mediated protection is accompanied by the upregulation of multiple sirtuin family members expressed in different cellular compartments, including Sirt1 & 2 (cytoplasm), Sirt3 & 5 (mitochon-dria), and Sirt7 (nucleus). Puncta of multiple sirtuins (Sirt1, 2, 3, and 5) were also ob-served in the ribbon synapse region. RT-qPCR confirmed the upregulation of Sirt3 but not other sirtuins at mRNA level. These findings highlight the potential of honokiol as a promising candidate for treating CIHL. Furthermore, sirtuins in different compart-ments form a cellular network for oxidative stress regulation. Sirtuin upregulation suggests their potential roles in honokiol-mediated hearing protection, which requires further investigation.”

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have satisfactorily addressed all the comments and implemented the necessary revisions to the manuscript. In its revised form, the manuscript is suitable for publication.

Author Response

Comments 1: The authors have satisfactorily addressed all the comments and implemented the necessary revisions to the manuscript. In its revised form, the manuscript is suitable for publication.

We greatly appreciate the comment of the reviewer.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have addressed most of the comments from my previous review, and I believe that the revised version is now much clearer. I only have a few comments regarding points that, in my opinion, could be further clarified. Addressing these issues would, I believe, help improve the presentation of the manuscript and make the results easier for readers to follow.

1. In my opinion, the information added about honokiol in the Introduction does not adequately address my previous comment. Although honokiol is a well-known compound to the authors, I believe that a considerable part of the scientific community may not be sufficiently familiar with this compound. Therefore, I suggested that the Introduction should include one or two sentences briefly introducing honokiol.

More specifically, the authors should indicate that honokiol is a compound belonging to the lignan group, mention its main natural sources, and briefly describe its relevant biological effects. This was also emphasized in my previous review, but it appears that my suggestion was interpreted differently. The purpose of this comment is not to request an extensive description of honokiol, but simply to provide sufficient background information for readers who may not be familiar with this compound.

2. The explanation added regarding the statistical analysis of the mean body weight is, in my opinion, not fully adequate. For this parameter, I believe that the figure itself should clearly indicate, using appropriate statistical significance symbols, whether the values for each experimental group (Cycles 1, 2, and 3) are statistically different from those of the Cis and HNK groups. This would allow the reader to immediately identify the statistically significant differences directly from the figure. Also, it also seems that the mean body weight for the HNK 10 + Cis (Cycle 1) group is not higher than that of the Cis (Cycle 1) control group, as might be expected from the current presentation.

In addition, I find that Figure 3B is not sufficiently clear in its current form. On the x-axis, two bars are labeled “HNK 10”, while, according to the colors indicated in the legend, these two bars represent values from Cycles 1 and 2. Based on the description of the experimental design, however, it appears that there are HNK 10 and HNK 20 groups, which subsequently undergo different cycles of cisplatin treatment.

Author Response

In my opinion, the information added about honokiol in the Introduction does not adequately address my previous comment. Although honokiol is a well-known compound to the authors, I believe that a considerable part of the scientific community may not be sufficiently familiar with this compound. Therefore, I suggested that the Introduction should include one or two sentences briefly introducing honokiol.

We greatly appreciate the suggestion and apologize for misinterpreting it in the first revision. We have revised the relevant text in the Introduction accordingly, as follows:

“We previously showed that Sirt3 was upregulated during hearing protection against cisplatin ototoxicity using honokiol [38] , a naturally occurring biphenolic lignan found in the root and bark of genus Magnolia [39]. Honokiol is a multifunctional molecule and a reported Sirt3 activator exhibiting a broad range of pharmacological activities, including anti-inflammatory, antioxidant, antitumor [40,41], and normal tissue protective effects [42-44]. It is an approved dietary supplement in Europe and was recently tested in combination with chemotherapeutic drugs to enhance anticancer efficacy [45-47].”

We also slightly adjusted relevant context in the Discussion.

The explanation added regarding the statistical analysis of the mean body weight is, in my opinion, not fully adequate. For this parameter, I believe that the figure itself should clearly indicate, using appropriate statistical significance symbols, whether the values for each experimental group (Cycles 1, 2, and 3) are statistically different from those of the Cis and HNK groups. This would allow the reader to immediately identify the statistically significant differences directly from the figure. Also, it also seems that the mean body weight for the HNK 10 + Cis (Cycle 1) group is not higher than that of the Cis (Cycle 1) control group, as might be expected from the current presentation.

Again, we greatly appreciate the suggestion and are particularly grateful to the reviewer for pointing out the error we made regarding the HNK20 group.

We have now marked the statistical significance for the relevant groups in Fig. 3B and revised the corresponding text accordingly, as follows:

“A following Games–Howell post-hoc testing showed that the weight losses were significantly lower in honokiol co-treated groups. Significant differences were detected in cycle 1 (p = 0.025 for HNK20+Cis group), Cycle 2 (p = 0.026 for HNK5+Cis group) and Cycle 3 (p = 0.024 and 0.028, for HNK5+Cis and HNK20+Cis groups, respectively) when compared to Cis-Only group (Fig. 3B).”

The reviewer is exactly right that the weight of the HNK10+Cis group is not higher than that of the Cis-Only group in any of the three cycles. This is likely due to the high variability of both the HNK10+Cis and Cis-Only groups.

Regarding the HNK20-Only group, we stated in Results 2.1 in the first revision: “Therefore, a group of female FVB wild-type mice treated with honokiol with a dose of 20 mg/kg/day (n=8) or vehicles (n=6) were used as the healthy control, which was also included in Fig. S1.” Since the HNK20-Only group were wild type animals rather than the tumor bearing mice, we did not include the data in Fig. 3. Instead, the weight changes of this group are presented in Supplementary Fig. S3.

For further clarification, we have now added the group name in the parentheses in the revised text: “(HNK20-Only, n=8)”.

We also corrected the error in Table 1, where the non-existing “Group #4, HNK20-Only” was listed in the MMTV-PyMT strain. This group has now been removed from Table 1. We sincerely apologize for this error and one more time greatly appreciate the reviewer for bringing it to our attention.