Proteomics Analysis of Antitumor Activity of Agrimonia pilosa Ledeb. in Human Oral Squamous Cell Carcinoma Cells

Round 1

Reviewer 1 Report

In this paper, Kim and colleagues investigated the use of proteomics and molecular biology approach to evaluate the antitumor effect of methanolic extract from roots of Agrimonia pilosa.

This is a potentially interesting paper, but it needs work to be considered for publication.

Overall, discussion must be improved, as well as the images quality.

Moreover, I have not found any supplemental material, although some are mentioned in the text

Line 82

Was the root dry? Fresh? Freeze-dried?

Line 89

What is ME-R? Please specify

Line 93

I understand that the lyophilized extracts were resuspended in DMSO. Was DMSO without extract used as control? It is not clear.

Line 108

Some fundamental data are missing. How were the proteins extracted? Have they been quantified? How?

How many ug of protein were loaded per gel?

Fig. 1C

To detect cell apoptosis, the cellular apoptotic proteins cleaved caspase 3 and cleaved-PARP were analyzed using western blot. Please add a graph with statistical analysis and specify the number of replicates.

Line 200

2D gel electrophoresis was performed to confirm the expression of proteins regulating apoptosis.

It is necessary to mark the considered spots on the representative gels. A list of identified differentially abundant proteins, including fold changes, must be included.

It seems that, in general, the total proteins are less abundant in gel 2 (treated samples). Was a normalization done during the spot analysis?

Moreover, what was used as a control?

Figure legend does not correctly report what the image represents.

Line 228- Fig. 4

Statistical differences are not highlighted in the graph and Std deviation is missing.

Moreover, how many replicas have been made?

Line 275-284

Please move this part into Materials and Methods

Conclusion

Authors claim that the pharmacological activity of Agrimonia pilosa may be attributed primarily to phenolic compounds (Line 259). Do the authors have any indication on the type of molecules responsible for the observed effects? I believe this would be a useful insight. The authors could investigate the nature of the molecules responsible for reported effects, for example, by precipitating and resuspending the protein part and then analyze its contribution.

Author Response

Line 82

Was the root dry? Fresh? Freeze-dried?

• Thank you for your comments and we were purchased dried roots.

Line 89

What is ME-R? Please specify

• Thank you for your comments, ME-R means lyophilized extract so we changed the words.

Line 93

I understand that the lyophilized extracts were resuspended in DMSO. Was DMSO without extract used as control? It is not clear.

• Thank you for your comments and DMSO without extract was used as control.

Line 108

Some fundamental data are missing. How were the proteins extracted? Have they been quantified? How?

How many ug of protein were loaded per gel?

• Thank you for your comments. Protein extraction and quantification were conducted by Yonsei Proteome Research Center. And 540μg of protein was loaded per gel.

Fig. 1C

To detect cell apoptosis, the cellular apoptotic proteins cleaved caspase 3 and cleaved-PARP were analyzed using western blot. Please add a graph with statistical analysis and specify the number of replicates.

• Thank you for your comments and we added graph.

Line 200

2D gel electrophoresis was performed to confirm the expression of proteins regulating apoptosis.

It is necessary to mark the considered spots on the representative gels. A list of identified differentially abundant proteins, including fold changes, must be included.

It seems that, in general, the total proteins are less abundant in gel 2 (treated samples). Was a normalization done during the spot analysis?

Moreover, what was used as a control?

Figure legend does not correctly report what the image represents.

• Thank you for your comments and we marked the spots and label the predicted protein labled, also.

Line 228- Fig. 4

Statistical differences are not highlighted in the graph and Std deviation is missing.

Moreover, how many replicas have been made?

• Thank you for your comments and we did three replicas, also STDEV added to graph.

Line 275-284

Please move this part into Materials and Methods

• Thank you for your comments and we considered your suggestion, but ‘Line 275-284’ is not method part. So we decided to leave it.

Conclusion

Authors claim that the pharmacological activity of Agrimonia pilosa may be attributed primarily to phenolic compounds (Line 259). Do the authors have any indication on the type of molecules responsible for the observed effects? I believe this would be a useful insight. The authors could investigate the nature of the molecules responsible for reported effects, for example, by precipitating and resuspending the protein part and then analyze its contribution.

• Thank you for your comments. In 2020, we published about APL study. According to previous study, diversity of phenolic compounds was detected.

Reviewer 2 Report

Abstract

The abstract contains a lot of information about the traditional use of Agrimonia pilosa, percentages of cancer incidence (can be found in the Introduction) and less data on methods, results and brief conclusion.

Introduction:

- Please specify what you wanted to say exactly in this sentence: "methanol extract from the root of APL, a protein altered by APL"

 - Usually, a plant name is abbreviated in this way: A. pilosa

 - Please fill in with some chemical data and information related to the antitumor action (what was done, the chemicals that would show anticancer action, mechanism, etc.), even if it was done at Discussions

2. Materials and methods OSCCs cell line - are they human?

2.2.Extraction: "APL roots were purchased from M&C (Changwon, Korea)". Please specify the company and who identified the plant / medicinal product?

ME-R and APL-M - fill in the name where it first appears in the text

Line 298 - begins with a comma.

Author Response

Answers to Reviewer-2

Abstract

The abstract contains a lot of information about the traditional use of Agrimonia pilosa, percentages of cancer incidence (can be found in the Introduction) and less data on methods, results and brief conclusion.

• Thank you for your comments. We edited the sentences as below.
  “Pharmacologically, A. pilosa has been reported about anti-inflammatory affection in Porphyromonas gingivalis LPS-induced RAW 264.7 cells [12], antitumor activity in lung cancer cell lines, breast cancer cell lines,  and prostate cancer cell lines through G0 state arrest [13], antiviral activity of SARS-CoV-2 [14], antioxidant activity through DPPH radical-scavenging activity and superoxide radical scavenging activity [15], an-tibacterial in methicillin-resistant Staphylococcus aureus [16], osteogenic differentiation acceleration in MC3T3-E1 cells by targeting miR-107 [17], blood coagulation function [18], and anti-hyperglycemic activity through inflammatory response inhibition in high-fat diet-fed rats [19], respectively.”

Introduction:

- Please specify what you wanted to say exactly in this sentence: "methanol extract from the root of APL, a protein altered by APL"

• Thank you for your comments. we modified the sentence as below.
  “In this study, we employed proteomics and molecular biological approach to examine the antitumor effect of methanol extract from the root of pilosa and a protein altered by A. pilosa extract, and to elucidate its mechanism of action.”

 - Usually, a plant name is abbreviated in this way: A. pilosa

• Thank you for your comments and we changed the abbreviation of Agrimonia pilosa to A. Pilosa.

 - Please fill in with some chemical data and information related to the antitumor action (what was done, the chemicals that would show anticancer action, mechanism, etc.), even if it was done at Discussions

• Thank you for your comments. So, we added following sentence. “According to MS/MS analysis, (2R,3R)-Taxifolin-3′-O-β-D-glucopyranside, (24S)-5α,8α-Epidioxy-ergost-6-en-3β-ol, 3-(4'-Hydroxy-benzyl)-5,7-dihydroxy-6,8-dimethyl-chroman-4-one, and (E)-Hexadecyl-ferulate are identified in APL and these components has anti-tumor effect.”

2. Materials and methods OSCCs cell line - are they human?

• Thank you for your comments. OSCCs cell line is human. We added that information.

2.2.Extraction: "APL roots were purchased from M&C (Changwon, Korea)". Please specify the company and who identified the plant / medicinal product?

ME-R and APL-M - fill in the name where it first appears in the text

• Thank you for your comments and we deleted ‘ME-R’ and changed APL-M to APL and added full name of it where it first appears.

Line 298 - begins with a comma.

• Thank your for your comments and we edited that sentence.

Reviewer 3 Report

This is an experimental work that aims to reveal a possible mechanism of action of Agrinomia pilosa root extracts on oral tumors.

According to the results obtained, the extracts of this plant do have anticancer capacity.

However, this work needs major corrections to be published.

The title must be changed, indicating that it is an in vitro test, which shows the anticancer effect of the plant based on proteomic and molecular studies.

This plant (Rosaceae) has a wide worldwide distribution and the reason for choosing it for this type of study is not well justified.

We do not doubt the reproducibility of the investigation, but there is no discussion, it only reproduces what has been said previously. Comparisons with similar research are lacking.

Nor does it make any kind of allusion to the biomolecules that could be involved in this antitumor action.

I believe that the work only describes some experiments and does not delve into anything else, and this could happen with many plants not yet studied

Author Response

This is an experimental work that aims to reveal a possible mechanism of action of Agrinomia pilosa root extracts on oral tumors.

According to the results obtained, the extracts of this plant do have anticancer capacity.

However, this work needs major corrections to be published.

The title must be changed, indicating that it is an in vitro test, which shows the anticancer effect of the plant based on proteomic and molecular studies.

• Thank you for your comments and we changed the title as below. “Proteomics analysis of anti-tumor activity of Agrimnoia Pilosa in human Oral squamous cell carcinoma cells”

This plant (Rosaceae) has a wide worldwide distribution and the reason for choosing it for this type of study is not well justified.

• Thank you for your comments and we added “ pilosa is a perennial plant that has been used clinically for a wide variety of maladies including hemostasis, antidiarrhea, detoxication and it heals and relieves parasitic infections. Our team revaled anti-inflammatory effect in RAW 264.7 cells and listed several anti-cancer components following MS/MS analysis. These results are the reasons why we tried to verify the anti-tumor effects in A.pilosa”. in introduction.

We do not doubt the reproducibility of the investigation, but there is no discussion, it only reproduces what has been said previously. Comparisons with similar research are lacking.

• Thank you for your comments. We already mentioned about studies about Pilosa Ledeb in discussion. Specially, there are no studies about “Human Oral squamous cell carcinoma”. So, we couldn’t compare our studies with other research. Please, understand generously..

Nor does it make any kind of allusion to the biomolecules that could be involved in this antitumor action.

• Thank you for your comments and the major compound of Pilosa, grilmonin was less effective than our extracts in human Oral squamous cell carcinoma. So, we didn’t mention our manuscript. This paper focused in proteomics to find anti-cancer activity proteins. This method is the first suggestion in human OSCC cell line by A. pilosa extracts. Next, studies will focus other fractions, hexane, chloroform, ethyl acetate, water, etc.

I believe that the work only describes some experiments and does not delve into anything else, and this could happen with many plants not yet studied

• Thank you for your comments and We already investigated Agrimonia Pliosa Ledeb and their MS/MS analysis in 2020. According previous our study, we revised our manuscript.

Round 2

Reviewer 1 Report

The authors have made some of the required changes but there are still unanswered questions and minor revisions to be made prior to publication. English doesn’t seem to have improved. 

Line 125

Authors claim that protein extraction and quantification were conducted by Yonsei Proteome Research Center but this is not enough. If there is no reference, the method used must be reported

Delete “and” at the beginning of the sentence. 

Line 226-229 

English needs to be revised.

Within the pH 200 range?? 

The list of identified DAPs (13+25) is still absent. I believe that this is an important data. Please add it as SM. 

There is still an unanswered question: Was a normalization done during the spot analysis?

Line 228

Delete “and” at the beginning of the sentence.

Line 311

“Several spots are matched peptides were heterogeneous”. I don't understand the meaning of the sentence. Please rewrite it.

Author Response

Thank you for your comments, our manuscript is improved a lot.

Line 125

Authors claim that protein extraction and quantification were conducted by Yonsei Proteome Research Center but this is not enough. If there is no reference, the method used must be reported.

→ Thank you for your comment. We edited and cited reference also.

Delete “and” at the beginning of the sentence.  

 → Thank you for your comment. We deleted it.

Line 226-229 

English needs to be revised.

 → Thank you for your comment.  We rivesed our manuscript, entirely.

Within the pH 200 range?? 

 → Thank you for your comment. It is pH 10. 

The list of identified DAPs (13+25) is still absent. I believe that this is an important data. Please add it as SM. 

→ Thank you for your comment. We edited our manuscript.

There were 48 spots with significant changes, but 13 protein spots were decreased and 11 increased spots were identified by MS/MS analysis. Identified proteins were listed and attached as supplymentary data.

There is still an unanswered question: Was a normalization done during the spot analysis? 

→ Yes, we did normalization. One of control sample used for normalization.

Line 228

Delete “and” at the beginning of the sentence.

→ Thank you for your comment. We deleted "and".

Line 311

“Several spots are matched peptides were heterogeneous”. I don't understand the meaning of the sentence. Please rewrite it.

→ Thank your for your comment. We rewrited that sentance. 

Reviewer 3 Report

The article may be published referring to the answers you just offered.

Author Response

Thank you for your comment.

We rewirted the manuscript and ask to English language editing through Editage.