Norcantharidin Enhances the Antitumor Effect of 5-Fluorouracil by Inducing Apoptosis of Cervical Cancer Cells: Network Pharmacology, Molecular Docking, and Experimental Validation

Round 1

Reviewer 1 Report

The manuscript presented by Huang et al. explores the synergistic anticancer effect of NCTD and 5-FU on two cervical cancer cell lines. The authors used a combination of in vitro experimental work, network pharmacology and molecular docking analysis to demonstrate that NCTD can potentiate the apoptotic effects of 5-FU, primarily through a caspase-dependent pathway. The methodological approach is robust, combining computational and experimental techniques. However, I consider that further clarification of some experimental details, and a more thorough discussion of the findings' implications would enhance the paper. Some observations are given below.

Major issues:

• How many biological replicates did you perform for your experimental assays? I don’t see any mention of this anywhere in the methodology section or figure captions except for blots.
• Line 52 – please source the information that NCTD is an active ingredient of Mylabris and other TCMs. I find this to be inaccurate, as cantharidin is the active ingredient in Mylabris, not NCTD, which is just synthetically produced (https://cmjournal.biomedcentral.com/articles/10.1186/s13020-020-00338-6). Otherwise, modify accordingly.
• Section 2.3. It is clear from the results that you used 24h, 48h and 72h incubation times. Please modify the methods section to include this information.  Also, please include information about what (treated), (blank) and (control) mean. Did you use PBS instead of CCK-8 solution for blank? Did you use an appropriate dilution for DMSO for control?
• For figure 1B, it would really help to have a log representation of the x-axis, with drug in molar or micromolar concentration. Your representation is a bit hard to follow. I can deduce 0 on the x axis means micromolar concentration, and 3 means millimolar, but it’s really not a common representation and can cause confusion. For an example of what I mean, see Figure 1 from the following article: https://www.nature.com/articles/s41420-022-01207-x . In addition, please use bigger points on your plots or different linetypes for the timepoints. I can barely understand which is which. Also, I find odd the viability increase at low NCTD concentrations for HeLa cells. Did you normalize to negative control?
• Section 2.9. Please provide the PDB IDs one the used protein structures.
• Line 183 – Please provide the IC50 values.
• Figure 2A-B. I’m not sure if there’s any statistical significance there. Did you perform any statistics? I’m not sure what 6-9 mean. Can you provide the combination concentrations in the caption?
• Line 224 – But you have positive FITC staining in HeLa cells from the 5-FU group. Please mention this.
• Lines 244-245. You mean the NOS3 and AHR targets were not available from the RCSB? Or that you couldn’t find binding sites on them? Please correct.
• Lines 248-252 need extensive rephrasing. The phrase ‘the hydrogen bond between the NCTD of Caspase-9 protein acted on two amino acid residues, viz. … etc.’ is not correct. Caspase-9 does not have NCTD. You can say ‘the hydrogen bonds formed between NCTD and Caspase-9 occurred between the … [insert atoms involved in hydrogen bonding from each molecule here]...’. Also, the 4.66Å distance, stated for ARG-180 and NCTD can barely be considered hydrogen bonding, as usually, a cut-off of 3.5Å is used when defining such an interaction. I see that this residue is relevant to your discussion, but from my point of view, it would be more likely that NCTD induces a conformational change that makes 5-FU binding more facile. This means that the two drugs most likely do not compete for binding to the same residue. I suggest you do a short Molecular Dynamics run (10 ns should be enough) starting from the best docking poses to see if the hydrogen bonds are kept throughout the simulations. This would make the amino-acid preference of each drug more clear and would make your discussion more solid. As is, lines 254-256 are too speculative. For an example methodology, see the following article: https://www.mdpi.com/1424-8247/16/6/865 .
• Line 266 – what do you mean by ‘are closely related to’? Do you mean ‘most likely bind to’?
• Lines 279-296 – I would merge this with the introduction part of your manuscript, as it's not really relevant to the discussion. I would, however, scout the literature to find if there’s any evidence of NCTD acting synergistically with any other anticancer drug.
• Lines 300-302 – How did you prove that toxicity is reduced? You do not show dose-response curves for drugs in combination, so we can’t know if IC50s are reduced.
• Line 308 – you didn’t show that the combination has a high binding affinity for Caspase-9. You only showed that both drugs have affinity for Caspase-9.
• The discussion would benefit from addressing the potential toxicity and side effects of a combined treatment. While enhancing apoptosis in cancer cells is desirable, it's also critical to consider the therapeutic window and safety profile of a combined treatment, especially if you address the applicability of a combined treatment in a clinical setting.

Minor issues:

• You probably omitted the other # from equal contribution. I'm guessing there should be two authors.
• Lines 62-70. Can you rephrase not to repetitively use the word ‘ultimately’?
• For scientific plant names, use italics e.g. Salvia miltiorrhiza Bunge
• Line 130 – adjust the number of cells seeded
• It would be great if you could provide undistorted images for the chemical representations of 5-FU and NCTD in Figure 1A.
• You should keep protein naming consistent throughout the manuscript: Caspase-3 and Caspase-9. Please recheck.

In particular, you should pay attention to time tenses. If you're stating something in general, you should use present tense e.g. line 59 should be: 'NCTD affects the expression of Caspase-9 and Caspase-3, proteins which induce apoptosis.'

Author Response

Dear professor：

        Thank you for your letter and the constructive comments on this article, that helped us enhance the quality of our manuscript.

         Please see the attachment for the revised manuscript and peer-to-peer reply at the end of file.

Sincerely,

Cheng-Cheng Wang

The Medical College of Guizhou University, Guizhou University (South Campus), Guiyang Province, China;

Tel: +86/15195873601;

E-mail: [email protected]

Author Response File: Author Response.pdf

Reviewer 2 Report

2. Abstract, line 12 you wrote that 5-Fluorouracil is the main chemotherapy drug in cervical cancer. According to current guidelines, it’s not true.

3. Give please some few details concerning Mylabris (line 52).

4. Line 53-54, you wrote that Norcantharidin has the advantages that is easy to synthesis. It would be interesting to add how it can be synthesized.

5. Materials and methods, please add:

- how much DMSO did you used to prepare the stock solutions

-which are the concentrations of Norcantharidin and 5-Fluorouracil used in the experimental study

6. Please improve the quality of Figures 3 and 4.

8. In introduction, lines 52-53 you specified that Norcantharidin has been successfully utilized as an effective anticancer drug in clinical practice. Give please more details at discussion subchapter (lines 293-95).

9. The discussions concerning the current results are brief. Please try to improve them.

10. Using the information presented at lines 325-334 please reformulate a conclusion. At the moment you first wrote “in summary” and then “conclusions”. In summary ≈ conclusions.

Minor editing of English language is required.

Author Response

Dear professor：

        Thank you for your letter and the constructive comments on this article, that helped us enhance the quality of our manuscript.

         Please see the attachment for the revised manuscript and peer-to-peer reply at the end of file.

Sincerely,

Cheng-Cheng Wang

The Medical College of Guizhou University, Guizhou University (South Campus), Guiyang Province, China;

Tel: +86/15195873601;

E-mail: [email protected]

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

I thank the authors for their reply. Most of my requests were fulfilled in the point-by-point response provided by authors, yet some aspects were not directly modified in the manuscript, and are only justified in the authors’ reply. For example, the authors did not provide the methodological details of the performed molecular dynamics simulations and should update their manuscript with this information, not just provide a reference for an article which has performed something similar.

I also identified some basic mistakes which I also pointed out in my previous report, such as faulty terminology use: for example, binding affinity should be used for the drug in relation to its target, and not vice versa (see line 339). Interestingly, the authors used this term correctly in the abstract and in lines 319 and 331 of the revised manuscript. Please be more consistent in using this term.

I would modify lines 256-259 as follows, in order to not put very much emphasis on the encountered error: ‘Molecular docking analysis assessed the potential binding of NCTD and 5-FU to the identified hub targets and their involvement in cell apoptosis, except NOS3 and AHR, for which a software error was encountered.’

The molecular dynamics results should be a bit more detailed, as the phrase “the Caspase-9-5-FU complex was more hydrogen-bonded and continuous, and the Caspase-9-NCTD complex was less hydrogen-bonded and unstable” lacks scientific soundness. You very beautifully responded to my comments in the author’s reply, but somehow did not include this information in the manuscript. Could you provide more information on the MD results in the Discussion part?

Lines 335-338: you should definitely discuss the limitations of this hypothesis, e.g. add a phrase like ‘but this should be further investigated through competitive binding assays’.

Can you add a more general conclusion to your work? Like what do you expect your work to bring to the scientific community?

Some extensive English language corrections should be made, especially in the newly modified parts. I recommend the use of some sort of English proofreading software such as Grammarly or LanguageTool, or reaching out to a company which provides proofreading services.

Author Response

Thank you for your constructive comments on this article, which helped us to improve the quality of our manuscript. We have carefully read your comments and discussed and revised each of them. For your convenience, we have combined the authors' responses to the reviewers and the manuscript into a single document. If any of the answers or questions in the manuscript are incorrect, please do not hesitate to let us know.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript has been significantly improved. The authors addressed all the required issues and in my opinion, the manuscript is suitable for publication.

Minor editing of English language is required.

Author Response

  Thank you for your constructive and valuable comments on this article, which helped us to improve the quality of our manuscript. We have carefully read your comments and discussed and revised the English language issues. For your convenience, we have combined the authors' responses to the reviewers and the manuscript into a single document.

  Thank you again for your input on our manuscript and we look forward to hearing from you.

Author Response File: Author Response.pdf