Ailanthoidol, a Neolignan, Suppresses TGF-β1-Induced HepG2 Hepatoblastoma Cell Progression
, Chien-Heng Shen 6,* and Hsing-Chun Kuo 7,8,9,10,*Round 1
Reviewer 1 Report
In Introduction the authors should add a part on characteristic, properties and classification of lignans and neolignans and related updated references should be added such as:
Pan, J.-Y.; Chen, S.-L.; Yang, M.-H.; Wu, J.; Sinkkonen, J.; Zou, K. An update on lignans: Natural products and synthesis. Nat. Prod. Rep. 2009, 26, 1251–1292.
Durazzo et al. Dietary Lignans: Definition, Description and Research Trends in Databases Development. Molecules. 2018 Dec 8;23(12):3251. doi: 10.3390/molecules23123251.
Durazzo et al. Lignan Content in Cereals, Buckwheat and Derived Foods. Foods. 2013 Feb 7;2(1):53-63. doi: 10.3390/foods2010053.
Teponno, R.B.; Kusari, S.; Spiteller, M. Recent advances in research on lignans and neolignans. Nat. Prod. Rep. 2016, 33, 1044–1092.
Kuhnle, G.G.C.; Dell’Aquila, C.; Aspinall, S.M.; Runswick, S.A.; Mullingan, A.A.; Bingham, S.A. Phytoestrogen content of foods of animal origin: Dairy products, eggs, meat, fish, and seafood. J. Agric. Food Chem. 2008, 56, 10099–10104.
A graphical scheme of study design should be inserted and more details of methodologies added.
The description of "3.1. The cytotoxicity of ATD on HepG2 hepatoblastoma cells. " should be implemented.
Data in Figures 3 and 4 should be better described in the text.
Lines 224-230 and 240-243 should be implemented.
Limits, advantages, applications and future directions should be added in Conclusion.
Author Response
Author's Reply to the Review Report (Reviewer 1):
Reviewer #1:
Comments and Suggestions for Authors
In Introduction the authors should add a part on characteristic, properties and classification of lignans and neolignans and related updated references should be added such as:
Pan, J.-Y.; Chen, S.-L.; Yang, M.-H.; Wu, J.; Sinkkonen, J.; Zou, K. An update on lignans: Natural products and synthesis. Nat. Prod. Rep. 2009, 26, 1251–1292.
Durazzo et al. Dietary Lignans: Definition, Description and Research Trends in Databases Development. Molecules. 2018 Dec 8;23(12):3251. doi: 10.3390/molecules23123251.
Durazzo et al. Lignan Content in Cereals, Buckwheat and Derived Foods. Foods. 2013 Feb 7;2(1):53-63. doi: 10.3390/foods2010053.
Teponno, R.B.; Kusari, S.; Spiteller, M. Recent advances in research on lignans and neolignans. Nat. Prod. Rep. 2016, 33, 1044–1092.
Kuhnle, G.G.C.; Dell’Aquila, C.; Aspinall, S.M.; Runswick, S.A.; Mullingan, A.A.; Bingham, S.A. Phytoestrogen content of foods of animal origin: Dairy products, eggs, meat, fish, and seafood. J. Agric. Food Chem. 2008, 56, 10099–10104.
Response: We appreciate this comment and we have added to the revised version of the introduction and related updated references in the manuscript.
A graphical scheme of study design should be inserted and more details of methodologies added.
Response: Thanks for the comments. These are described that flow chart of ATD administered after initial of TGFb1 pretreated HepG2 in fig 1, line 91 to line 92 in the text.
The description of "3.1. The cytotoxicity of ATD on HepG2 hepatoblastoma cells. " should be implemented.
Data in Figures 3 and 4 should be better described in the text.
Lines 224-230 and 240-243 should be implemented.
Limits, advantages, applications and future directions should be added in Conclusion.
Response:
We appreciate this comment and we have added to the revised version of the discussion and the full text of manuscript from page 5 line 195 to line 201, page 6 line 216 to line 221, page 11 line 374 to line 402.
Author Response File: 
Author Response.doc
Reviewer 2 Report
The authors presented in their study a novel mechanism on that ATD exerts anticancer potential via suppression cell progression of TGF-β1-promoted HepG2 cells while involving blocking of p38MAPK and Smad 2 signaling.
abstract: what is "anti-progression" stated in the abstract? the word is not very often used
the introduction is very short, not introducing the stated problem in bigger detail, it should be improved
methods - what grade of FBS was it?
why the authors used antibiotics for cell cultivation? that is simply wrong and it can significantly affect the results
regarding antibodies - the exact type/clone must be stated, ideally the catalogue number of the antibodies, since as we know, the reproducibility with antibodies might be very bad since it depends on the exact type of antibody used
preparation of ATD stock solution is missing - solvent, concentration, storage, stability...
cytotoxicity assay - the number of replicates is totally missing, also, what was used as a positive and negative control, data evaluation is missing as well as the statistics, etc.
moreover, why only 24 and 48 h was used, the most often 72 h is used to see the longer effect
line 116 - correct: 5 x 105
cells/well - this unit does not exist ! did you mean cells per well?
line 120 - CO2 - 2 should be in subscript
wound healing assay - SD as three independent experiments is wrong since the authors wrote that they examined three fields of view in one sample, so how many biological replicates were done in reality?
what type of matrigel was used?
correct paraformaldehydel for paraformaldehyde, in reality, it is formaldehyde and only after the fixation/polymerization, it is paraformaldehyde
crystal violet - manufacturer? in what it was dissolved??
3 fields of view are definitely not sufficient, it should be at least 10, and also biological replicates must be done, not only fields of view in one sample, you can simply not do any statistics from that
colony assay - replicates? quantified by Image J how? the description is totally missing; crystal violet - what form, what percentage, solvent, etc.
"separated by SDS-polyacrylamide gels and electrophoretically transferred to the PVDF" - describe in detail, conditions of the experiment are fully missing
dilutions of the antibodies missing, the total volume of the antibody missing, imager used missing, exposure time missing...
results: chapter: The cytotoxicity of ATD on HepG2 hepatoblastoma cell
there is almost none description of the results, the few sentences mostly repeat the methodology, describe better and in more detail the results
the same for wound healing assay - the results should be described in more detail, the same for other chapters
the discussion is very poor and must be extended
Figure 9 is very small and pixelated, it is very hard to read
the legend to the figure should be more descriptive of the mechanism depicted in the Figure
remove the ref. to Figure 9 from the conclusion
the conclusion could be more informative
Author Response
RE: Revised version of biomedicines-1339828
Dear Mr. Franklin Zhou
Assistant Editor, M.Sc.,
Enclosed please find one revised version entitled: Ailanthoidol, a neolignan, suppresses TGF-β1-induced HepG2 hepatoblastoma cell progression, which we would like to submit for publication in Biomedicines.
This revised version has been carefully corrected according editor and referee’s reports point-by-point. We appreciate these valuable comments to strengthen our presentation. Please inform me if any revision is needed. The file marked change in blue color.
Furthermore, I would verify that no part of the manuscript is under consideration for publication elsewhere and it will not submit elsewhere if accepted by Biomedicines and not before the Editorial Office has reached a decision.
Sincerely yours,
Hsing-Chun Kuo, Ph.D.
Professor,
Institute of Nursing and Department of Nursing,
Chang Gung University of Science and Technology,
Chia-Yi Campus, Taiwan.
E-mail: guscsi@gmail.com
TEL: +886-5-3628800
FAX: +886-5-3628866
Author's Reply to the Review Report (Reviewer 2):
Comments and Suggestions for Authors
The authors presented in their study a novel mechanism on that ATD exerts anticancer potential via suppression cell progression of TGF-β1-promoted HepG2 cells while involving blocking of p38MAPK and Smad 2 signaling.
abstract: what is "anti-progression" stated in the abstract? the word is not very often used
Response: Thanks for the comments. The words are replaced by "against progression of liver cancer" in page 1, line 42 to line 43 in the text. The error in manuscript were revised in blue color.
the introduction is very short, not introducing the stated problem in bigger detail, it should be improved
Response:
We appreciate this comment and we have added to the revised version of the conclusion and the full text of manuscript from page 2 line 61 to line 63, line 72 to line 89.
methods - what grade of FBS was it?
why the authors used antibiotics for cell cultivation? that is simply wrong and it can significantly affect the results
regarding antibodies - the exact type/clone must be stated, ideally the catalogue number of the antibodies, since as we know, the reproducibility with antibodies might be very bad since it depends on the exact type of antibody used
Response: We appreciate this remark of comment and the materials was revised in manuscript from page 3 line 95 to line 109.
preparation of ATD stock solution is missing - solvent, concentration, storage, stability...
Response: ATD was dissolved in DMSO (stock solution: 100 mM) and put in brown bottle, then stored in the freezer before experiment was revised in manuscript from page 4 line118.
cytotoxicity assay - the number of replicates is totally missing, also, what was used as a positive and negative control, data evaluation is missing as well as the statistics, etc.
moreover, why only 24 and 48 h was used, the most often 72 h is used to see the longer effect
Response: We appreciate this comment and we have added to the revised version of 2.9 Statistical analysis in manuscript from page 5 line187 to line 190.
Data were expressed as mean ± S.D. (n = 6/group) of three independent experiments and were analysed by one-way ANOVA. The data were analysed using the SAS software statistical package ‘SigmaPlot’, version 9.0 (SAS Institute Inc., Cary, NC, USA).
The cytotoxic effect of ATD was evaluated to the HepG2 hepatoblastoma cells for 24 and 48 hand and then 0-50 μM concentrations of ATD were determined the molecular mechanism of ATD inhibition associated with TGFb1 pretreated HepG2 cell progression in this study.
The cytotoxicity assay 50 μM mitomycin c as positive control and solvent 0.02% DMSO, as negative control was used before.
line 116 - correct: 5 x 105
cells/well - this unit does not exist ! did you mean cells per well?
line 120 - CO2 - 2 should be in subscript
Response: Thanks for the comments. line 129 - correct: Cells (5 x 105 cells per well), line 133 - correct: 5% CO2.
wound healing assay - SD as three independent experiments is wrong since the authors wrote that they examined three fields of view in one sample, so how many biological replicates were done in reality?
Response: We appreciate this comment and we have added to the revised version of 2.9 Statistical analysis. The invasion cells was quantified in 6 randomly selected fields per well (n = 6) using a light microscope and data were expressed as mean ± S.D. of three independent experiments. We have added to the revised version of 2.9 Statistical analysis in manuscript from page 5 line187 to line 190, page 7 line245 to line 246.
what type of matrigel was used?
Response: We appreciate this remark of comment and the materials was revised in manuscript from page 3 to line 105 to line 108. Matrigel is a solubilized basement membrane preparation extracted from murine sarcomas, and contains proteoglycans, type IV collagen, laminin, and growth factors (Catalog Number: 356234 BD Biosciences, Bedford, MA, USA).
correct paraformaldehydel for paraformaldehyde, in reality, it is formaldehyde and only after the fixation/polymerization, it is paraformaldehyde
Response: Thanks for the comments. Line 148 -Correct: paraformaldehyde.
crystal violet - manufacturer? in what it was dissolved??
3 fields of view are definitely not sufficient, it should be at least 10, and also biological replicates must be done, not only fields of view in one sample, you can simply not do any statistics from that
Response: Thanks for the comments. 0.1% crystal violet in ethanol were purchased from Sigma Chemical Co. (St. Louis, MO, USA). We have added to the revised version of 2.9 Statistical analysis. The cells was quantified in 6 randomly selected fields per well (n = 6) using a light microscope and data were expressed as mean ± S.D. of three independent experiments. We have added to the revised version of 2.9 Statistical analysis in manuscript from page 5 line187 to line 190, page 7 line245 to line 246.
colony assay - replicates? quantified by Image J how? the description is totally missing; crystal violet - what form, what percentage, solvent, etc.
Response: Thanks for the comments and the methods was revised in manuscript from page 4 line 155 to line 160.
"separated by SDS-polyacrylamide gels and electrophoretically transferred to the PVDF" - describe in detail, conditions of the experiment are fully missing
dilutions of the antibodies missing, the total volume of the antibody missing, imager used missing, exposure time missing...
Response: Thanks for the comments and the methods was revised in manuscript from page 5 line 172 to line 185.
Proteins were then blotted onto NC membranes (Sartorious), and reacted with the primary antibodies (integrin α3 (1:10000), vimentin (1:10000), N-cadherin(1:2000), MMP2(1:2000), Smad 2/3 (1:2000), and tubulin (1:10000) were obtained from Santa Cruz Biotechnology, Inc., CA., USA. Anti-p-p38MAPK (1:1000), anti-p38MAPK (1:1000), and anti-p-Smad 2/3 (1:2000) were from Cell Signaling Technology (Beverly, MA, USA)). The secondary antibody was a peroxidase-conjugated goat anti-mouse antibody (1:10000), obtained from Santa Cruz Biotechnology, Inc., CA., USA. After binding, the bands 1 hour were revealed by enhanced chemiluminescence using the ECL commercial kit.
results: chapter: The cytotoxicity of ATD on HepG2 hepatoblastoma cell
there is almost none description of the results, the few sentences mostly repeat the methodology, describe better and in more detail the results
Response: Thanks for the comments and the result 3.1 was revised in manuscript from page 5 line 195 to age 15 to line 201.
the same for wound healing assay - the results should be described in more detail, the same for other chapters
Response:
We appreciate this comment and we have added to the revised version of the figure 3 detail in manuscript from page 8 line 249 to line 257.
the discussion is very poor and must be extended
Response:
We appreciate this comment and we have added to the revised version of the discussion and the full text of manuscript from page 11 to line 374 to line 398.
Figure 9 is very small and pixelated, it is very hard to read
the legend to the figure should be more descriptive of the mechanism depicted in the Figure
Response:
We appreciate this comment and we have added to the revised version of the Figure 9 legend from page 12 to line 399 to line 402.
Author Response File: 
Author Response.pdf
Round 2
Reviewer 2 Report
The authors have thoroughly answered all questions, added the required information, statistics, and all that was asked, therefore, now, I can recommend the article to be accepted.
