A Bee Trp-Arg Dense Peptide with Antiproliferation Efficacy against the Prostate Cancer Cell Line DU145

Round 1

Reviewer 1 Report

A very modest antiproliferative activity of two short WR-rich peptides derived from bee. The amount of data presented is limited (no control, one unique cell line) and the end of the Introduction does not clearly enounce the objective of the study.

Major points:

1.       The two studied peptides derive from Tritrpticin. Therefore, this peptide should be used as a control, otherwise the work presents little interest.

2.       Formulation and solution stability of the peptides? Are the peptides stable in solution and in the cell culture medium? Which type of formulation was used?

3.       Anti-proliferative action. Fig 1 shows data after 24hrs of cell culture. Usually, anti-proliferative tests are conducted with cells cultivated from 48 and 72hrs. Can you provide data after at least 48 hours, including also a control drug (as a reference).

4.       The spheroid model in interesting but the peptide effect is very weak. Here also, a reference product (control) is missing (anticancer drug or Tritrpticin-type peptide).

5.       The apoptosis data in Fig 4 are not convincing at all. The peptide-induced effect is minimal, hardly visible. Moreover, Fig 4 is not clearly labelled (PI/annexin V). Better evidenced for apoptosis should be presented (caspase activation, sub-G1 profile, …).

6.       The authors claim that the peptide induce S/G2 phase cell cycle arrest. Where are the data? Cell cycle profile (subG1/G1/S/G2) should be presented.

7.       Section 2.2. cannot be entitled « Synthesis Trp-Arg dense peptide » without detailing the synthesis. The peptides were simply bought from a vendor company. The synthetic procedure should be mentioned (liquid phase or solid support?) and importantly, the formulation used to test the peptides must be given.

8.       There is absolutely no evidence to support the claim that the peptide can interfere with ion channel in the cell membrane, causing Ca2+ influx, This is a very speculative idea. It may (?) be proposed in the discussion but certainly not cited in the abstract.

Specific points:

-          The sequence of the two studied short peptides N0820 and N0821 should be indicated in the abstract.

-          Table 1. A brief presentation of the four bee species (A. cerana, A. dorsata, A.mellifera, A. florea) would be useful. Indicate below the table what are these species?

Altogether, the manuscript is not very solid. An extensive revision is required.

OK

Author Response

We thank the editors and the reviewers for their thoughtful and helpful comments. We have addressed, in a point by point manner, all the suggestions and queries from the reviewer and marked them with red in the manuscript. The input from the reviewers has allowed us to improve the clarity and quality of our paper. We have included below our point by point response to the reviewers’ comments and have included these additions and alterations to the revised manuscript.

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

I recommend attaching the images of the spheroids

It is not clear what the statistical analysis is, what the data are and what they express. A breakdown of the analysis.

Authors to define N0820 and N0821

The conclusions reflect the recorded results to a small extent. They must be relevant both for the specialist and for people interested in the subject.

Author Response

We thank the editors and the reviewers for their thoughtful and helpful comments. We have addressed, in a point by point manner, all the suggestions and queries from the reviewer and marked them with red in the manuscript. The input from the reviewers has allowed us to improve the clarity and quality of our paper. We have included below our point by point response to the reviewers’ comments and have included these additions and alterations to the revised manuscript.

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The revised manuscript is only slightly improved. The (very limited) data remain weakly convincing. The peptide displays a weak anti-proliferative action against one cell line, not an "anticancer efficacy". The title is scientifically erroneous.

Author Response

We thank the editors and the reviewers for their thoughtful and helpful comments. I extend my sincere gratitude for your thorough review of my paper. Your insightful comment and keen interest have significantly contributed to the refinement of the manuscript.

Please see the attachment.

Author Response File: Author Response.pdf

Round 3

Reviewer 1 Report

The revised manuscript is slightly improved. OK.

Author Response

Thank you for your thoughtful review. Thanks to your feedback, my manuscript was able to develop further.