Ribitoborate Synergism with Histone Deacetylase Inhibitor Romidepsin as a Potential Treatment for Breast Cancer

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The work “Ribitoborate Synergism with Histone Deacetylase Inhibitor Romidepsin as a Potential Treatment for Breast Cancer” is interestingly, however, several achievements should be done before publication.

The Western-blot are from different membranes, therefore, can not be used to compare the expression of proteins. From the shape and form it can be observed that Bcl-2 and Cyclin D; Survivin and c-Myc; p21, H3K27 and H3K9; H3 and Actin were incubated in the same membrane, therefore the membrane were antibodies for Bcl-2 and Cyclin D were assayed should be incubated with Actin. The same procedure should be done for Survivin and c-Myc and p21, H3K27 and H3K9. If you have the images of its respective Actin expression, please include them.

It is necessary to improve the quality of the propidium iodide (PI)/Hoechst 33342 staining images; the resolution is so low that neither condensation nor fragmentation can be observed.

It is necessary to improve the quality of the immunocytochemical images; the resolution is so low that cannot be observed.

Author Response

Dear Editorial Board Members, IJMS

We would like to thank our peer-reviewed for the comments and for their useful suggestions made to help us strengthen the manuscript presented, and appreciate the opportunity given to address any concerns raised. Below is our formal response to each of the reviewer’s points to accompany the revised manuscript with additional experiments performed, and text modified per the suggestions received.

We thank the reviewer for taking the time to read and analyze our manuscript.

Reviewer 1:

Comments and Suggestions for Authors

The work “Ribitoborate Synergism with Histone Deacetylase Inhibitor Romidepsin as a Potential Treatment for Breast Cancer” is interestingly, however, several achievements should be done before publication.

Comment: The western blots are from different membranes, therefore, cannot be used to compare the expression of proteins. From the shape and form it can be observed that Bcl-2 and Cyclin D; Survivin and c-Myc; p21, H3K27 and H3K9; H3 and Actin were incubated in the same membrane, therefore the membrane were antibodies for Bcl-2 and Cyclin D were assayed should be incubated with Actin. The same procedure should be done for Survivin and c-Myc and p21, H3K27 and H3K9. If you have the images of its respective Actin expression, please include them.

Response: We appreciate the Reviewer’s point. We used equal amount protein estimated by Bradford assay and this is stated in Materials and Methods section. The representative β-actin housekeeping protein expression is the further confirmation of equal amount loading in all groups of different treatments. Therefore, we provided one β-actin expression to represent. We did multiple times showing the expression was same in all time points among different treatment groups.

 

Comment: It is necessary to improve the quality of the propidium iodide (PI)/Hoechst 33342 staining images; the resolution is so low that neither condensation nor fragmentation can be observed.

Response: We would like to thank the reviewer for the suggestion. According to Reviewer’s suggestion, we have replaced the better-quality images of Figure 4A & 4B.

 

Comment: It is necessary to improve the quality of the immunocytochemical images; the resolution is so low that cannot be observed.

Response: We appreciate the reviewer’s comments. According to Reviewer’s suggestion, we have replaced the better-quality images of Figure 6A & 6B. We would also remind the reviewer that the original images are of higher resolution which might not be directly viewed within the pdf file.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This article describes the combination therapy of boron-containing ribitol and histone deacetylase inhibitors (HDAC) against triple-negative breast cancer. The idea is novel and the design is reasonable, however after reading the manuscript, the following concerns and suggestions have arisen.

The most important doubts:

1. To determine the type of interaction between ribitoborate and HDAC inhibitors, the Authors should use the coefficients described in the literature, for example, the combination index (Cancers 2020, 12(8), 2196; https://doi.org/10.3390/cancers12082196, Medical Oncology (2026) 43:113, https://doi.org/10.1007/s12032-025-03213-2).
2. What solvent was used to prepare the ribitoborate and HDAC inhibitors solution? What was the highest solvent concentration in the single- and two-component systems?
3. Figure 4: PI is commonly used to detect dead cells, but to identify apoptosis (and distinguish it from e.g. necrosis) it is necessary to use other dyes at the same time (most often annexin V). It would be optimal to perform a flow cytometric analysis
4. Figure 5: Western blot results should be supplemented with quantitative densitometric analysis.

Minor errors:

1. I suggest moving the graphs with EC50 calculations to supplementary materials, and placing the EC50 values ​​on the graphs of Figure 1 (I) or in the additional table.
2. Please improve the readability of the concentration values ​​in Figure 2 (B).

 

Author Response

Dear Editorial Board Members, IJMS

We would like to thank our peer-reviewed for the comments and for their useful suggestions made to help us strengthen the manuscript presented, and appreciate the opportunity given to address any concerns raised. Below is our formal response to each of the reviewer’s points to accompany the revised manuscript with additional experiments performed, and text modified per the suggestions received.

We thank the reviewer for taking the time to read and analyze our manuscript.

Reviewer 2:

Comments and Suggestions for Authors

This article describes the combination therapy of boron-containing ribitol and histone deacetylase inhibitors (HDAC) against triple-negative breast cancer. The idea is novel and the design is reasonable, however after reading the manuscript, the following concerns and suggestions have arisen.

The most important doubts:

1. To determine the type of interaction between ribitoborate and HDAC inhibitors, the Authors should use the coefficients described in the literature, for example, the combination index (Cancers 2020, 12(8), 2196; https://doi.org/10.3390/cancers12082196, Medical Oncology (2026) 43:113, https://doi.org/10.1007/s12032-025-03213-2).

Response: We completely appreciate the reviewer’s point. The combination index (CI) was calculated for the analysis of the synergistic, antagonistic or additive effects of the two drugs ribitoborate and romidepsin. The CI was calculated using the formula: C=[(D)1/(Dx)1] + [(D)2/(Dx)2], in which (D)1 was the concentration of the first drug required to achieve a particular effect in the combination; (Dx)1 was the concentration of the first drug that causes an identical effect alone; (D)2 was the concentration of the second drug that achieves a particular effect in the combination; (Dx)2 was the concentration of the second drug that generates the same effect alone. CI>1 indicates antagonism, CI=1 indicates an additive effect and CI<1 indicates synergy. We found that the synergistic effect was closely dependent on romidepsin concentrations. Indeed, for all ribitoborate concentrations tested, synergism was observed for romidepsin concentrations at 0.5nM (Table S1).

These analyses presented in the main text page 4 and highlighted in yellow confirmed the synergistic action of the ribitoborate and romidepsin combination. We have added a paragraph in the materials and methods section (page 12, highlighted in yellow), explaining how we have proceeded to conduct these analyses, and provided table in supplementary section as Table S1.

 

Comment: What solvent was used to prepare the ribitoborate and HDAC inhibitors solution? What was the highest solvent concentration in the single- and two-component systems?

Response: For ribitoborate, we used water to make stock solutions and for HDAC inhibitors we used DMSO to make stock solutions. The method we prepared ribitoborate was described in the Materials and Methods section. We purchased HDAC inhibitors from sellekchem (10mM). The stocks were further diluted in culture medium at least 500 times (<0.1% DMSO) as stated in the Material and Methods section (page 11).

 

Comment: Figure 4: PI is commonly used to detect dead cells, but to identify apoptosis (and distinguish it from e.g. necrosis) it is necessary to use other dyes at the same time (most often annexin V). It would be optimal to perform a flow cytometric analysis.

Response: Thank you, the reviewer for the valid point. We agree with the reviewer that Annexin V is a generally accepted marker of apoptosis. Our studies used Propidium iodide (PI) and Hoeschst primarily to examine the death of TNBC cells. PI, a DNA-binding dye, is excluded by intact cell membranes but penetrates late apoptotic cells. Early apoptotic cells exclude PI, while late-stage apoptotic cells stain positive for both Annexin V and PI. This result is therefore suggestive of, although does not validate death by apoptosis. We have now modified the description accordingly. 

1. Figure 5: Western blot results should be supplemented with quantitative densitometric analysis.

Response: Thank you for reviewer’s suggestion. We have provided densitometric analysis of western blot data supplemented as figure S2

Minor errors:

1. I suggest moving the graphs with EC50 calculations to supplementary materials and placing the EC50 values ​​on the graphs of Figure 1 (I) or in the additional table.

Response: According to reviewer’s suggestion, we have mentioned EC50 values on respective graph in figure 1. Also, we moved those EC50 graphs to supplementary section S1.

1. Please improve the readability of the concentration values ​​in Figure 2 (B).

Response: Thank you for reviewer suggestion. We agree with the reviewer’s point. As per reviewer suggestion, now we have provided better quality image of readout.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript investigates the potentially synergistic effects of ribitoborate and the histone deacetylase (HDAC) inhibitor romidepsin in triple-negative breast cancer (TNBC). While identifying a novel combination therapy to lower the effective dose of HDAC inhibitors is an interesting iidea, the current manuscript contains overstatements that must be addressed.

Entire study relies exclusively on a single TNBC cell line, MDA-MB-231. Despite this severe limitation, the authors make sweeping claims that the drug combination has great potential for the "treatment of TNBC" and should be explored as a therapy for "other solid cancers". To validate these claims, the authors must test the combination on a diverse panel of TNBC cell lines, as well as on normal human breast epithelial cells, to prove broad efficacy and confirm selective cytotoxicity.

The authors state that the combination of romidepsin and ribitoborate "holds promise for future clinical applications in TNBC therapy". Making translational clinical assertions based entirely on basic in vitro cell viability and migration assays is a major scientific flaw. The study entirely lacks in vivo animal models, which are strictly necessary to evaluate the systemic toxicity, pharmacokinetics, and actual tumor-shrinking efficacy of this unproven drug combination.

In the methods section, the authors state they evaluated differences between groups using a "student’s t-test for unpaired observations". Using a standard t-test to compare multiple experimental groups (e.g., Control, Ribitoborate alone, Romidepsin alone, and Combination) is a fundamental statistical issue that dramatically inflates the risk of false-positive (Type I) errors. The authors must re-analyze their multi-group data using an appropriate Analysis of Variance (ANOVA) with post-hoc corrections.

The authors attempt to explain the synergy by showing western blots of common apoptosis and cell cycle markers (such as p53, p21, Bcl-2, H3k9, and H3k27). However, this data only shows correlative downstream effects of cell death rather than uncovering the actual synergistic mechanism between the two drugs. The authors themselves openly admit in the discussion that "the role boron plays in the alteration of gene expression and cell death is not clear," which entirely undermines their mechanistic conclusions.

The authors must correct their statistical methods, rewrite their clinical claims, and provide additional cell line and in vivo data to prove the safety and efficacy of this treatment.

 

Author Response

Dear Editorial Board Members, IJMS

We would like to thank our peer-reviewed for the comments and for their useful suggestions made to help us strengthen the manuscript presented, and appreciate the opportunity given to address any concerns raised. Below is our formal response to each of the reviewer’s points to accompany the revised manuscript with additional experiments performed, and text modified per the suggestions received.

We thank the reviewer for taking the time to read our manuscript and for making constructive remarks.

Reviewer 3:

Comments and Suggestions for Authors

The manuscript investigates the potentially synergistic effects of ribitoborate and the histone deacetylase (HDAC) inhibitor romidepsin in triple-negative breast cancer (TNBC). While identifying a novel combination therapy to lower the effective dose of HDAC inhibitors is an interesting idea, the current manuscript contains overstatements that must be addressed.

Comment: Entire study relies exclusively on a single TNBC cell line, MDA-MB-231. Despite this severe limitation, the authors make sweeping claims that the drug combination has great potential for the "treatment of TNBC" and should be explored as a therapy for "other solid cancers". To validate these claims, the authors must test the combination on a diverse panel of TNBC cell lines, as well as on normal human breast epithelial cells, to prove broad efficacy and confirm selective cytotoxicity.

Response: We agree with the reviewer that understanding the difference in response to the ribitoborate and romidepsin combination across multiple cell lines could also provide valuable knowledge for future studies and its target population as almost all anti-cancer drugs have limited target cancer population.  We consider this study is an initial one and much more studies are required to determine its applicability in human and its target cancer population. Nevertheless, we modified the description and conclusion to reflect this fact. Specifically, we modified the statements that “drug combination has great potential for the "treatment of TNBC" and should be explored as a therapy for "other solid cancers" to “The underlying molecular mechanisms for synergy could be further explored for potential development of new combined therapy for breast cancer” (page 1).

We agree with the reviewer that it is important to know which sub-population of human cancer would benefit from this combined therapy. We plan to extend our study to other breast cell lines to define markers of responsiveness. 

Comment: The authors state that the combination of romidepsin and ribitoborate "holds promise for future clinical applications in TNBC therapy". Making translational clinical assertions based entirely on basic in vitro cell viability and migration assays is a major scientific flaw. The study entirely lacks in vivo animal models, which are strictly necessary to evaluate the systemic toxicity, pharmacokinetics, and actual tumor-shrinking efficacy of this unproven drug combination.

Response: We would like to thank the reviewer for the suggestion. We completely agree with the reviewer’s comments. Our overall aim was to establish proof of concept that the combination could be more effective than each drug administrated separately. This seems important to us before spending major efforts to optimize the combination therapy to apply in vivo. In our laboratory, 3-D culture studies are going on and exploring the mechanism of action in detail to check the therapeutic activity of ribitoborate and romidepsin. We are planning to conduct an in-depth efficacy study, including in vivo once the combination is optimized. We will look forward to being able to present these results in subsequent manuscripts.

We have replaced the sentence "holds promise for future clinical applications in TNBC therapy” by “Further study could elucidate the detail mechanisms of action and potentially leads to development of better drug combination for breast cancer” (Page 2), to tune down our conclusion and avoid possible misinterpretation.

Comment: In the methods section, the authors state they evaluated differences between groups using a "student’s t-test for unpaired observations". Using a standard t-test to compare multiple experimental groups (e.g., Control, Ribitoborate alone, Romidepsin alone, and Combination) is a fundamental statistical issue that dramatically inflates the risk of false-positive (Type I) errors. The authors must re-analyze their multi-group data using an appropriate Analysis of Variance (ANOVA) with post-hoc corrections.

Response: This is a very valid point. We agree with the reviewer and re-analyze multi-group data using an Analysis of Variance (ANOVA) with post-hoc corrections. Comparisons were carried out using one-way analysis of variance with post-hoc corrections. We have now clarified that point in the Material and Methods section (Page 13) as “The significance of difference in treatment groups was determined using one-way ANOVA with- post-hoc corrections, where value of p<0.05 between the groups was considered as statistically significant difference between these groups. 

Comment: The authors attempt to explain the synergy by showing western blots of common apoptosis and cell cycle markers (such as p53, p21, Bcl-2, H3k9, and H3k27). However, this data only shows correlative downstream effects of cell death rather than uncovering the actual synergistic mechanism between the two drugs. The authors themselves openly admit in the discussion that "the role boron plays in the alteration of gene expression and cell death is not clear," which entirely undermines their mechanistic conclusions.

Response: To our knowledge, this is the first study reporting the combination of ribitoborate with romidepsin against breast cancer. We agree that it is a very interesting point that we plan to analyze in further studies, using a pharmacologically optimized compound. We plan 1) to analyze the effect of the optimized combination on various TNBC cells and 2) to perform the reviewer's suggested specific mechanism of action of ribitoborate and romidepsin combination. We are continuing to explore this mechanism of action in detail in subsequent studies and look forward to being able to present these results in subsequent manuscripts.

We understand the importance of establishing a mechanism for the observed synergy of ribitoborate and romidepsin, but we also realize the complexity of the network. In agreement with the reviewer, experiments are underway to exploring more signaling molecules within the network in cell culture and in vivo with the aim to establish more detailed mechanism of action. We therefore consider these further studies beyond the current manuscript. Clearly much needs to be studied for its mechanism of action and potential applications in vivo and in clinics.

This study tested ribitoborate as an individual compound. Individual element could be investigated but will not change the biological effect of ribitoborate as a defined compound.

 

Comment: The authors must correct their statistical methods, rewrite their clinical claims, and provide additional cell line and in vivo data to prove the safety and efficacy of this treatment.

Response: Thank you for Reviewer’s suggestions which are important for new drug development. We however understand that it is much too early to determine the safety and efficacy in this early study. We are in fact continuing this study as the reviewer advised 

We thank the reviewer for his constructive comments, and we have revised our manuscript accordingly. We have addressed all the reviewers’ comments.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors precisely as you mentioned in your respond “ We used equal amount protein estimated by Bradford assay and this is stated in Materials and Methods section. The representative β-actin housekeeping protein expression is the further confirmation of equal amount loading in all groups of different treatments. Therefore, we provided one β-actin expression to represent. We did multiple times showing the expression was same in all time points among different treatment groups.” You have several blots showing that the expression was the same in all time points among different treatment groups, therefore, I request, at a minimum, the β-actin expression data corresponding to the blots included in the manuscript. It is incorrect to assert the expression levels of proteins analyzed on different gels and membranes; to evaluate the expression of an experimental protein, the expression of the reference (housekeeping) protein must be visualized on the same membrane.

Author Response

Dear authors precisely as you mentioned in your respond “We used equal amount protein estimated by Bradford assay and this is stated in Materials and Methods section. The representative β-actin housekeeping protein expression is the further confirmation of equal amount loading in all groups of different treatments. Therefore, we provided one β-actin expression to represent. We did multiple times showing the expression was same in all time points among different treatment groups.” You have several blots showing that the expression was the same in all time points among different treatment groups, therefore, I request, at a minimum, the β-actin expression data corresponding to the blots included in the manuscript. It is incorrect to assert the expression levels of proteins analyzed on different gels and membranes; to evaluate the expression of an experimental protein, the expression of the reference (housekeeping) protein must be visualized on the same membrane.

Response: We appreciate the reviewer for taking the time to read our manuscript and for making constructive remarks. We agree with the reviewer that we should always run a house keeping expression along with the protein of interest on same membrane. Here, we are providing western blots of the β-actin along with the protein of interest ran on the same time with same lysate using equal concentration of protein. We apologize that we did not do it β-actin on the same membrane of protein of interest, but did do 3 of them, which are now inserted into the figure. We hope this plus the fact that we use of same amount of lysate from the same sample should be satisfactory.    

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The Authors have incorporated most of the earlier comments; however, a few corrections still require clarification.

Please provide the reference for the method used to calculate CI. Minor error: In the Materials and Methods section, the equation uses „C" instead of „CI”.

Statistical analysis of the densitometric results is necessary to discuss differences in the expression of pro- and anti-apoptotic genes between the treated cells and the control. It is possible that, following statistical analysis, it will be necessary to modify the description provided in the paragraph: „Effect of romidepsin and ribitoborate on survival and apoptotic proteins”.

 

 

 

Comments for author File: Comments.pdf

Author Response

Reviewer 2

The Authors have incorporated most of the earlier comments; however, a few corrections still require clarification.

Please provide the reference for the method used to calculate CI. Minor error:In the Materials and Methods section, the equation uses „C instead of „CI”.

Statistical analysis of the densitometric results is necessary to discuss differences in the expression of pro- and anti-apoptotic genes between the treated cells and the control. It is possible that, following statistical analysis, it will be necessary to modify the description provided in the paragraph: „Effect of romidepsin and ribitoborate on survival and apoptotic proteins”.

 

Response: We appreciate the reviewer for taking the time to read our manuscript and for making constructive remarks. We are sorry for typographical errors and incorporated statistical analysis for western blot densitometric results. We have revised our manuscript accordingly (Highlighted in text as yellow color). We have addressed all the reviewer’s comments.

Here are the edits/modifications in revised manuscript:

1. We provided the reference for the method used to calculate CI, mentioned in revised manuscript. Please see page#12 under Materials and Methods section 4.3. Cell viability assay and synergy software and under references section, page #16, reference#48.
2. Yes, we corrected our typographical error, please see page#12 under Materials and Methods section 4.3. Cell viability assay and synergy software.
3. Now, we have provided statistical analysis of densitometric results and incorporated these results in revised manuscript under results section “Effect of romidepsin and ribitoborate on survival and apoptotic proteins”, page#6. Also, we incorporated the statistical analysis for the figure S2, please see supplementary file(s), figure S2, page#3.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The authors should resolve typographical errors in the discussion where "TRBC" is mistakenly written instead of "TNBC", and in Section 4.5 where "Hoechst 3032" is written instead of "Hoechst 33342". Additionally, there are minor inconsistencies regarding the reported single-agent romidepsin values, which vary between 0.94 nM, 0.945 nM, and 0.925 nM across different sections of the manuscript. Finally, the spelling of the assay "CellTiter-Glo" should be standardized throughout the text, as it currently appears in various forms including "Cell titer Glo", "CellTiter-Glow", and "CellTiter-Glo"

Author Response

Reviewer 3

The authors should resolve typographical errors in the discussion where "TRBC" is mistakenly written instead of "TNBC", and in Section 4.5 where "Hoechst 3032" is written instead of "Hoechst 33342". Additionally, there are minor inconsistencies regarding the reported single agent romidepsin values, which vary between 0.94 nM, 0.945 nM, and 0.925 nM across different sections of the manuscript. Finally, the spelling of the assay "CellTiter-Glo" should be standardized throughout the text, as it currently appears in various forms including "Cell titer Glo", "CellTiter-Glow", and "CellTiter-Glo"

 

Response: We thank the reviewer for taking the time to read our manuscript. We are sorry for typographical errors and now authors maintained uniformity in revised manuscript. We have revised our manuscript accordingly (Highlighted in text as yellow color). We have addressed all the reviewers’ comments.

Author Response File: Author Response.pdf