Epigallocatechin-3-gallate Synergistically Enhanced Arecoline-Induced Cytotoxicity by Redirecting Cycle Arrest to Apoptosis
, Po-Chi Hsu 2,†, Chih-Pin Chuu 3, Hao-Ai Shui 4,*,‡, Chien-Chih Yeh 1,‡, Yueh-Chung Chen 5,‡ and Yung-Hsi Kao 2,*,‡
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe aim of the manuscript was to determine whether the antioxidant EGCG contained in green tea could synergistically enhance the cytotoxic effects of the carcinogenic arecoline on cancer cells. The aim of the work was well defined by the authors and the research methods were appropriately selected. The discussion contains an important discussion of the research problem.
Below I present the main comments regarding the reviewed manuscript.
1. Introduction.
The introduction should be expanded with a more complete description of the properties of EGCG, which have multidirectional effects.
You can use, among others: from work published in MDPI https://doi.org/10.3390/antiox11081566,
2.Methods.
2.1. Cell Culture.
Please complete the company from which you purchased the cell culture dishes, culture media and the name of the C02 incubator used. Please correct the spelling of CO2.
There is also no information about the purity and source of the compounds used, i.e. EGCG and arecoline.
2.2. Cell Treatment and Viability Assay.
The concentrations of the compounds in the combination (EGCG and arecoline) and the type of microplate reader and company should be completed.
There is no information about the company from which the reagents were purchased (MTT, DMSO, etc.). This note applies to the entire methods section of the manuscript.
2.4. Reactive Oxygen Species Analysis.
Please complete the spectrophotometer type. There is a lack of concentration and solvent for NAC.
I propose to supplement Figure 2 with fluorescence staining using DCFDA and measuring the relative fluorescence intensity.
3. Results.
In order to fully visualize the level of apoptosis, Figure 3 should be supplemented with cytometric analysis (anexin V test). Additionally, fluorescent staining of cell nuclei using DAPI fluorochrome may be considered.
In Figure 8, part number 9 is difficult to read.
There is no completed part of the manuscript, i.e. Author Contributions.
Literature should be prepared in accordance with the journal's guidelines.
In summary, the manuscript is interesting but requires revisions before being reconsidered for publication in CIMB.
Author Response
We deeply appreciate your valuable suggestions on our manuscript. We have sincerely and carefully revised our manuscript based on your comments. Our detailed responses to your comments are included in the uploaded file. To clearly indicate the revisions, all revised sentences are highlighted in red in the revised manuscript. We hope our revisions address your concerns within the given limitations. Thank you for your understanding and consideration.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsPlease refer to attachment.
Comments for author File: Comments.pdf
Author Response
We deeply appreciate your valuable suggestions on our manuscript. We have sincerely and carefully revised our manuscript based on your comments. Our detailed responses to your comments are included in the uploaded file. To clearly indicate the revisions, all revised sentences are highlighted in red in the revised manuscript. We hope our revisions address your concerns within the given limitations. Thank you for your understanding and consideration.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsRemarks to authors:
In this manuscript, authors have reported that the epigallocatechin-3-gallate can efficiently decrease the intracellular ROS levels which were elevated by arecoline. Also, authors have demonstrated that the epigallocatechin-3-gallate induced cell-cycle arrest and facilitated cell apoptosis in arecoline-treated cells. That’s interesting, however, there are some suggestions for the manuscript.
--In this manuscript, authors demonstrated the results using the PC-3 cells, however, only one cell line might be not enough to support authors’ conclusion in this manuscript.
--In the introduction section, authors should provide more supporting information about the epigallocatechin-3-gallate.
Comments on the Quality of English LanguageThere are some minor mistake in English writing that needs some modifications.
Author Response
We deeply appreciate your valuable suggestions on our manuscript. We have sincerely and carefully revised our manuscript based on your comments. Our detailed responses to your comments are included in the uploaded file. To clearly indicate the revisions, all revised sentences are highlighted in red in the revised manuscript. We hope our revisions address your concerns within the given limitations. Thank you for your understanding and consideration.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsBelow are my comments regarding the reviewed manuscript titled: „Epigallocatechin-3-gallate reduced reactive oxygen species but synergistically enhanced arecoline-induced cytotoxicity through redirecting cycle arrest to apoptosis”.
1.Methods.
There is no information from which company the antibiotics and serum were purchased (line 82-83).
Thank you for explaining the limitations of the research. However, quantitative analysis of the level of apoptosis is still lacking. It seems to me that staining cells with annexin V is a very basic and inexpensive test that can be performed to analyze the level of apoptosis. This test not only identifies the early (annexin V-FITC (+) and dead cell marker (–)), but also the late stage of apoptosis (annexin V-FITC (+) and dead cell marker (+)). Hence, the results obtained from the proposed test would fully correlate with other results presented in the manuscript.
Research should not be limited by the argument that the journal has a strict 10-day publication deadline. When considering the level of a journal, you should look at the quality of research submitted to the journal, not the speed of publication.
From section 2.2. (Cell Treatment and Viability Assay), I propose to remove a concentration of 0 µM of the tested compounds. 0 µM=control.
2. Supplementary materials.
The supplemental materials include Figure 2, which is referred to in the manuscript as Figure 1A and B (line 211 and 215). I have doubts whether it is justified to transfer only one Figure to the supplementary materials, especially since it concerns the analysis of the ROS level, which is important from the point of view of the research purpose formulated by the authors and the title of the manuscript.
3. The authors still have not completed all the information in accordance with the manuscript template, in the following order:
-Supplementary Materials
-Author Contributions
-Funding
-Institutional Review Board Statement
-Informed Consent Statement
-Data Availability Statement
-Acknowledgments
-Conflicts of Interest
4. Discussion.
I suggest that the following sentence be removed from the discussion: "Future studies should include cytometric analysis, such as annexin V assay, to visualize early events of apoptosis."
As I have already mentioned, the cytometric test with annexin V is one of the basic tests in the analysis of cytotoxicity and apoptosis level, hence it should not be a stage of continuation and extension of research.
In summary, the manuscript is interesting but still has some limitations before publication in CIMB.
Author Response
Dear Reviewer,
We are profoundly grateful for your insightful suggestions and the opportunity to refine our manuscript further. Following your valuable feedback, we have undertaken a second round of careful and detailed revisions. Our responses to each of your comments from this latest review are thoroughly documented in the attached file.
To ensure clarity and ease of review, we have maintained the original revisions highlighted in red to signify changes made after the first set of comments. For the latest modifications stemming from your second round of feedback, we have highlighted these sentences in blue in the second revised manuscript.
The precise locations of these latest revisions (highlighted in blue) are clearly indicated, including the specific page and line numbers, to facilitate a straightforward review process.
We hope that these latest revisions comprehensively address your concerns and meet the high standards of the journal. We sincerely thank you for your continued understanding and consideration as we work together to enhance the quality of our manuscript.
Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsIn Line 105, change Choose to the Choice .......
Comments on the Quality of English LanguageNone, minor.
Author Response
Dear Reviewer,
We are profoundly grateful for your insightful suggestions and the opportunity to refine our manuscript further. Following your valuable feedback, we have undertaken a second round of careful and detailed revisions. Our responses to each of your comments from this latest review are thoroughly documented in the attached file.
To ensure clarity and ease of review, we have maintained the original revisions highlighted in red to signify changes made after the first set of comments. For the latest modifications stemming from your second round of feedback, we have highlighted these sentences in blue in the second revised manuscript.
The precise locations of these latest revisions (highlighted in blue) are clearly indicated, including the specific page and line numbers, to facilitate a straightforward review process.
We hope that these latest revisions comprehensively address your concerns and meet the high standards of the journal. We sincerely thank you for your continued understanding and consideration as we work together to enhance the quality of our manuscript.
Author Response File: Author Response.pdf
Round 3
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors complied with most of the reviewer's comments. The current version of the manuscript requires minor corrections. Comments are below.
1. Abstract. I suggest writing: Prostate cancer cells (PC3 cell line) instead of: Prostate cancer cell line PC3 cells.
2.Methods.
Point 2.8 is missing. Statistical analysis. It was not specified what statistical tests and software were used to analyze the obtained results.
3.Results.
Figure 2C lacks a scale or information in the figure caption at what magnification the observation was made. I suggest enlarging the photos from the fluorescence microscope and placing the graphs (quantitative analysis of Annexin V-FITC and PI staining) below. This will improve the quality of photos added to the manuscript.
In the description of Figures 2-6, please complete the concentrations of the tested compounds. They are only found in the description of Figure 1 and Figure 1 (Supplementary materials).
Figure 3A. On a cell cycle diagram, the phases should be placed in the following order: G1, S, G2/M.
4. Other comments: Spelling should be corrected (line: 150, 152).
Summary, the manuscript requires minor modifications before its publication, but a certain limitation of the research, as mentioned by the authors in the discussion, is the use of one cell line.
Author Response
Dear reviewer #1,
We extend our deepest gratitude for your constructive feedback on our manuscript. Detailed responses to each point have been carefully prepared and are included in the uploaded file.
We have detailed the specific locations of the 3rd revisions (highlighted in blue), including exact page and line numbers, to ensure these changes are easily identifiable.
We are thankful for your comments on improving our manuscript.
Author Response File: Author Response.pdf
