Therapeutic Effects of Cephalotaxus harringtonia Leaf Extract on Hepatocellular Carcinoma via Regulation of the Intrinsic Apoptosis Pathway and Cell Cycle
, Sonny C. Ramos 1,†, Hyun Bo Sim 1, Ju-Bin Lee 1, Ho-Yeol Jang 2, Beom-Gyun Jeong 2, Kyung-Wuk Park 2, Kyung-Yun Kang 2,3,* and Jong-Jin Kim 1,*Round 1
Reviewer 1 Report
Comments and Suggestions for Authors1. The detection time of membrane potential is 48 hours after medicine action, while other indicators are detected simultaneously at 24 and 48 hours. Please supplement the examination of changes in membrane potential after 24 hours of drug action to facilitate synchronous comparison of the timeliness of the mechanism of action.
2. In apoptosis research, the ratio of BAX/BCL-2 is considered a more reliable and sensitive indicator of apoptosis induction than a single protein concentration. Suggest supplementing the detection of BCL-2 protein expression.
3. The intrinsic apoptosis pathway and cell cycle are both mentioned in the title of the paper, indicating that apoptosis and cell cycle changes are equally important mechanisms of action. However, there is a lack of examination of related regulatory proteins in the cell cycle. Please supplement the detection of cycle related proteins based on the degree of cycle arrest.
4. In Figure 2AB, the concentration unit of CHLE is incorrect, it should not be μg, but μg/ml.
Author Response
|
Response to Reviewer 1 Comments
|
||
|
1. Summary
|
|
|
|
Dear Reviewer:
This is in reference with regards to the revision of our manuscript (Manuscript ID: cimb-3969652) titled “Therapeutic Effects of Cephalotaxus harringtonia Leaf Extract on Hepatocellular Carcinoma via Regulation of the Intrinsic Apoptosis Pathway and Cell Cycle”.
We would like to take this opportunity to express our sincere thanks to our dear reviewer who identified areas of our manuscript that needed corrections or modifications. We would also like to thank you for allowing us to resubmit a revised copy of the manuscript. Kindly check the corrections and response to your query.
We hope you will review the manuscript and take the necessary steps for its publication.
Sincerely yours,
Jong-Jin Kim Department of Biomedical Science Sunchon National University
|
||
|
2. Point-by-point response to Comments and Suggestions for Authors
|
||
|
Comments 1: The detection time of membrane potential is 48 hours after medicine action, while other indicators are detected simultaneously at 24 and 48 hours. Please supplement the examination of changes in membrane potential after 24 hours of drug action to facilitate synchronous comparison of the timeliness of the mechanism of action. Response 1: We thank the reviewer for this valuable suggestion. We have added the assessment of changes in membrane potential after 24 h of drug treatment to allow a synchronous comparison of the timeliness of the mechanism of action. At 24 h, mitochondrial membrane potential was reduced by more than 35% in the CHLE 10 µg/mL group, and this reduction became more pronounced at 48 h. The results are presented in Figure 5B. Response 2: We thank the reviewer for the insightful comment. As suggested, we have supplemented the detection of BCL-2 protein expression, which was presented in Supplementary Figure S4. In the 10 µg/mL CHLE group, BCL-2 expression significantly increased compared to the DMSO group (Figure S4A). However, the BAX/BCL-2 ratio increased over time (Figure S4B), indicating a stronger apoptotic signal. This data was already incorporated into the result part of the manuscript (lines 326-328).
Response 3: We thank the reviewer for this important comment. We have supplemented the requested data based on the observed degree of cycle arrest, and the results are presented in Supplementary Figure S3. CHLE inhibited the G1 phase in HepG2 cells, thereby reducing the S phase. Accordingly, we measured the mRNA expression levels of the cell cycle regulators cyclin E1 and CDK2, which control the G1–S phase transition. The results showed decreased expression of both regulators, as shown in Figure S3 (Lines 287-290). The qRT-PCR method was also included in the methodology (Lines 161-175).
Response 4: We thank the reviewer for the significant observation, and we apologize for the oversight and appreciate the opportunity to make these corrections. As you noted, we have corrected the concentration units from μg to μg/mL. The updated figures are as follows: Figures 2A-B, 3C, 4A-C, Figure S1, and Figure S2.
|
||
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsI have attached my comment.
Reviewer’s Comment
1. I advise that the authors add what initiated the extraction method or technique of the C. harringtonia leaf extract; how does this extraction method relate to the traditional use of the materials in healing.
2. In the methodology, the authors stated that acid was added to acetonitrile as well as to water in the HPLC-ELSD/MS analysis. I think it was a typographical error, and it needs to be corrected.
3. In the result section, it is noticed that a high percentage of S-phase at 162% was recorded and G2-phase 136% recorded (lines 254 and 255respectively). Since the total value of all phases should not exceed 100%. This should be looked at or explained.
Author Response
|
Response to Reviewer 2 Comments
|
||
|
1. Summary
|
|
|
|
Dear Reviewer:
This is in reference with regards to the revision of our manuscript (Manuscript ID: cimb-3969652) titled “Therapeutic Effects of Cephalotaxus harringtonia Leaf Extract on Hepatocellular Carcinoma via Regulation of the Intrinsic Apoptosis Pathway and Cell Cycle”.
We would like to take this opportunity to express our sincere thanks to our dear reviewer who identified areas of our manuscript that needed corrections or modifications. We would also like to thank you for allowing us to resubmit a revised copy of the manuscript. Kindly check the corrections and response to your query.
We hope you will review the manuscript and take the necessary steps for its publication.
Sincerely yours,
Jong-Jin Kim Department of Biomedical Science Sunchon National University
|
||
|
2. Point-by-point response to Comments and Suggestions for Authors
|
||
Comments 1: I advise that the authors add what initiated the extraction method or technique of the C. harringtonia leaf extract; how does this extraction method relate to the traditional use of the materials in healing.
Response 1: We thank the reviewer for this insightful comment. In this study, we employed a traditional hot-water extraction method, which is widely used in conventional medicinal preparations. This approach reflects the traditional practice of utilizing C. harringtonia leaves in herbal remedies. Moreover, by adopting a well-established extraction technique, we aimed to ensure the reproducibility and broader applicability of the extract for future research and potential therapeutic use.
Comments 2: In the methodology, the authors stated that acid was added to acetonitrile as well as to water in the HPLC-ELSD/MS analysis. I think it was a typographical error, and it needs to be corrected.
Response 2: We apologize for any confusion and appreciate the opportunity to clarify this point. In our HPLC-ELSD/MS analysis, acid was intentionally added to both the acetonitrile and water phases. Because a gradient elution system was used, it was necessary to maintain a constant acid concentration throughout the analysis. Therefore, the same concentration of acid was added to both mobile phases to ensure consistent ionization efficiency and stable chromatographic performance.
Comments 3: In the result section, it is noticed that a high percentage of S-phase at 162% was recorded and G2-phase 136% recorded (lines 254 and 255 respectively). Since the total value of all phases should not exceed 100%. This should be looked at or explained.
Response 3: We apologize for any confusion and appreciate the opportunity to clarify this point.
The percentages exceeded 100% because they were calculated relative to the corresponding cell-cycle phase of the DMSO control group. For example, if the S phase in the DMSO group was 13.7% and the S phase in the 10 μg/mL CHLE group was 22.2%, the ratio of 22.2% / 13.7% results in 162% (Figure 2D).
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe paper has been improved after the revision and can be accepted in its present form.
Author Response
|
Response to Reviewer 1 Comments
|
||
|
1. Summary |
|
|
|
Dear Reviewer:
This is in reference with regards to the revision of our manuscript (Manuscript ID: cimb-3969652) titled “Therapeutic Effects of Cephalotaxus harringtonia Leaf Extract on Hepatocellular Carcinoma via Regulation of the Intrinsic Apoptosis Pathway and Cell Cycle”.
We would like to take this opportunity to express our sincere gratitude to our dear reviewer, who identified areas of our manuscript that required corrections or modifications.
We would also like to thank you for reviewing our manuscript and recommending it for publication.
Sincerely yours,
Jong-Jin Kim Department of Biomedical Science Sunchon National University
|
||
|
2. Point-by-point response to Comments and Suggestions for Authors
Comments and Suggestions for Authors The paper has been improved after the revision and can be accepted in its present form.
Response: Thank you for your constructive insights, which contributed to the publication of our manuscript. |
||
|
|
||
