Novel Insights of Herbal Remedy into NSCLC Suppression through Inducing Diverse Cell Death Pathways via Affecting Multiple Mediators

Round 1

Reviewer 1 Report

The paper by Batbold and Liu 
entitled “Novel insights of herbal into tumor suppression through in- 
ducing diverse cell death pathways via affecting multiple mediators” 
explores the prospective antitumor effects of Artemisia santolinifolia extract (ASE) against two non-small cell lung cancer (NSCLC) cell lines, A549 and H23) and and their molecular mechanisms of action.

The authors found that ASE induces death in both cell lines with different mechanisms: apoptosis in H23 and ferroptosis in A549. The two death pathways are investigated in the correct way, analyzing the key markers of both the initiation and execution phases of the death processes.

 

General considerations

The work, written in a clear and fluent way, is convincing for some aspects analyzed, but it is much less so for others, which therefore should be clarified.

 

Major points

1. In the Graphical abstract the authors should insert the name of the cell line to which each of the two pathways refers (i.e. Caspase 3 cleavage or Lipid peroxidation)
2. The SRB assay does not provide a measure of proliferation at all, but rather of cytotoxicity. Authors must then correct this, starting with the paragraph title (4. Cell Proliferation Assay)
3. I find the morphological observations not correctly interpreted. There are no morphological characteristics that can distinguish the different types of death. For example, cellular rounding is also characteristic of apoptotic cells and not, as the authors argue, of cells during ferroptosis. This aspect could be clarified only through ultrastructural analysis with a transmission electron microscope, but not with optical microscope analysis.
4. As a specific ferroptosis inhibitor was used (DFO). In Figure 3D, the authors should also show data obtained on both lines using a specific apoptosis inhibitor, e.g. ZVAD.
5. How was cell death quantified in Figure 3D? The caption only indicates that it is a cytofluporimetric measurement, but not with what type of marking (annxin? Calcein? Trypan blue, Propidium iodide? Other?). Please specify.
6. Also I would have a curiosity. To what extent can the composition of plant extracts be standardized in terms of quantity of individual components? This would seem to be one of the main problems related to the use of natural substances.

Comments for author File: Comments.docx

Author Response

Dear Editor and Reviewer,

    We sincerely appreciate all valuable comments and suggestions of the Reviewers’ concerning our manuscript entitled “applsci-1674264”. We have carefully considered the comments and tried our best to address every one of them accordingly. The main corrections in the paper and our responses to all the comments are as follows:

 

Major points

1. In the Graphical abstract the authors should insert the name of the cell line to which each of the two pathways refers (i.e. Caspase 3 cleavage or Lipid peroxidation)

We appreciate for your comment. The bottom section of the graphical abstract was modified and the cell line names were indicated in accordance with the cell death modes they were more susceptible to, as well as distinct changes involved in cell death pathways, such as activation of caspase3, GPX4 inhibition, ROS, lipid peroxidation were linked in interpretation with corresponding cell lines.

2. The SRB assay does not provide a measure of proliferation at all, but rather of cytotoxicity. Authors must then correct this, starting with the paragraph title (4. Cell Proliferation Assay)

Thank you for your kind notice. We have corrected the paragraph title “2.4. Cell Proliferation Assay” to 2.4. “Cell Cytotoxicity Assay”.

3. I find the morphological observations not correctly interpreted. There are no morphological characteristics that can distinguish the different types of death. For example, cellular rounding is also characteristic of apoptotic cells and not, as the authors argue, of cells during ferroptosis. This aspect could be clarified only through ultrastructural analysis with a transmission electron microscope, but not with optical microscope analysis.

We totally agree with the referee's comment. The exact mechanisms underlying the phenotypic changes that occur during ferroptosis remain unclear; however, it is earlier have been reported to proceed morphologically distinct changes from other forms of cell death. The cells following treatment with a pro-ferroptotic agent, an initial cell shrinking is followed by condensation of cytoplasmic constituents and a “ballooning” phenotype, which involves the formation of a clear, rounded morphology consisting mainly of empty cytosol. In accordance with the referees’ comment, we have included previously reported study figures representing highly similar morphological features observed in our study. We would consider utilizing ultrastructural analysis with a transmission electron microscope in our future works. Also, in the result section describing the morphological changes, we have made some modifications to more distinct morphological features we observed in two cell lines.

 (Redox Biology, 2019)

4. As a specific ferroptosis inhibitor was used (DFO). In Figure 3D, the authors should also show data obtained on both lines using a specific apoptosis inhibitor, e.g. ZVAD.

Thank you for your suggestion. We would consider this aspect of using a specific apoptosis inhibitor in future studies.

5. How was cell death quantified in Figure 3D? The caption only indicates that it is a cytoflowmetric measurement, but not with what type of marking (annxin? Calcein? Trypan blue, Propidium iodide? Other?). Please specify.

Thank you for your suggestion. We have made an additional modification in Figure 3 legend and corresponding Results section to specify the flow cytometry measurement.

6. Also I would have a curiosity. To what extent can the composition of plant extracts be standardized in terms of quantity of individual components? This would seem to be one of the main problems related to the use of natural substances.

Thank you for addressing this point. We totally agree that identifying and quantifying the individual active component is the main problem related to the use of natural products. Initially, in our study, we aimed to use whole plant extract to show possible anticancer effects in NSCLC. Accordingly, no other separation techniques of compounds were used to identify different fractions of ASE. We have utilized only one kind of extract for compound identification. For this reason, we couldn’t predict for sure our findings may represent the sustainable source of active components in A.santolinifolia. In this stage, we could only predict the possibility of some active components possessing antiproliferative properties.

Author Response File: Author Response.pdf

Reviewer 2 Report

Overall this manuscript presents a generally well-designed study with notable impact in the field of cancer drug discovery, revealing multiple mechanisms of cellular death on ASE treatment. The manuscript, although well-written and referenced in the methods and discussion, could use further editing and clarification, particularly in sections such as the abstract and results. My major comment would be that the study lacks data on the mechanisms of migration or proliferation in the presence of ASE, however this is touched on by the authors in discussion and may be out of the scope of this journal, or currently under investigation for preparation in another manuscript? Other comments below.

Title

• Poor title. Incomplete and could be more specific.

Abstract

• Lacks introductory/background information.
• Results are described, but inconsistently and needs to be more focused. Reads too much like a discussion throughout the whole abstract. A lot of unnecessary text.
• Follow the format background/intro and aims > methods > results (or mixed methods/results) > conclusions and significance.
• Graphical abstract – Good basis for graphical abstract, the bottom section however could be adjusted to help the reader understand what they are looking at or how it should be read/interpreted and make a link between the top section (ASE) and it’s use in cells as that’s not clear in the Figure.

Intro

• Introduction is relatively short but to the point, which is fine.
• You mention 5-year survival rate has increased overall despite it still being low, but don’t mention the causes for this increase. Is the increase due to work in this field or other general developments in health/medicine? Don’t necessarily need to mention that it has increased as you want to focus on the significance of this work which is that cancer survival is too low and needs improving.
• The section on pharmacological drugs is rather broad and doesn’t seem cancer-specific, should add in some further examples.
• Careful of punctuation throughout.
• Last ‘paragraph’ of the introduction needs to be broken up into sentences. I also don’t mind using a results-driven descriptive section at the end of the intro, but it should at least be supported by a brief description of what the study aimed to do.

Methods

• Materials and methods relatively thorough
• Avoid repeating abbreviations that have been mentioned in the introduction i.e. NRF2, STAT3 etc.
• For antibodies and cell lines, you should consider adding RRIDs, although not essential if not requested by journal.
• Page 3 line 114 - subscript on CO₂
• Avoid swapping subheadings between title case and sentence case.
• ‘Cells were trypsinized’ – using trypsin-EDTA? What %? Or Trypsin alternative. Important information given expression of cell surface receptors and molecules will be impacted by trypsin.
• TBS buffer abbreviated without full term.

Results

Overall figures (in figure legends) need to indicate sample size where not showing representative images. In particular, histograms of Western blot data are described as ‘representative histograms’, but still have error bars and are used for statistical analysis, which should not be done on representative samples, only where data has been pooled from independent biological experiments. Therefore I am unsure if the authors truly mean the histograms are ‘representative’ or if they mean the Western blot images are ‘representative’ and histograms are data analysed from independent replicates?

• Figure 1 – Figures 1A and 1B x-axis should be µg/mL? Figure 1B “concentration” is misspelled on the x-axis. Should it not be “ASE concentration” on the X-axis in line with the text (not AS)?
• Authors chose to use the 72 hour IC₅₀ values, but later incubated these concentrations with cells for 24-48 hours. Can authors comment more specifically on why this was chosen or if there were statistical differences between the IC₅₀ for different times?
• Figure 2 – DTX (positive control) abbreviation not defined.
• Figure 3 – 3A seems unnecessary and doesn’t provide any additional information that isn’t presented in 3B, especially as the gated area does not appear to align with the measured area? 3B Y-axis is cut off on H23 graph. Also as above, sample size should be indicated as these histograms can’t be ‘representative’ as they have error and statistical analysis associated with them. In 3D, H23 control cells appear to have quite a high and variable cell death in the control cells, is there a reason for this?

 

Author Response

Dear Editor and Reviewer,

We sincerely appreciate all valuable comments and suggestions of the Reviewers’ concerning our manuscript entitled “applsci-1674264”. We have carefully considered the comments and tried our best to address every one of them accordingly. The main corrections in the paper and our responses to all the comments are as follows:

1. Overall this manuscript presents a generally well-designed study with notable impact in the field of cancer drug discovery, revealing multiple mechanisms of cellular death on ASE treatment. The manuscript, although well-written and referenced in the methods and discussion, could use further editing and clarification, particularly in sections such as the abstract and results. My major comment would be that the study lacks data on the mechanisms of migration or proliferation in the presence of ASE, however this is touched on by the authors in discussion and may be out of the scope of this journal, or currently under investigation for preparation in another manuscript? Other comments below.

Thank you for addressing this point. Initially, in our study, we aimed to use whole plant extract to show the possible anti-tumor effect in NSCLC. Accordingly, no other techniques were used to identify the effect of ASE on EMT properties of NSCLC. We have utilized two kind of parenteral cell lines in order to observe the cytotoxicity mechanism of ASE. For this reason, in this stage, we could only show the possibility of ASE possessing anticancer property in NSCLC through triggering distinct cell death pathways.

Title

2. Poor title. Incomplete and could be more specific.

Thank you for your comment. The title was modified to “Novel insights of herbal remedy into NSCLC suppression through inducing diverse cell death pathways via affecting multiple mediators”

Abstract

3. Lacks introductory/background information.

We appreciate for your comment, the sentence containing background information was added in the abstract section. 

4. Results are described, but inconsistently and needs to be more focused. Reads too much like a discussion throughout the whole abstract. A lot of unnecessary text.

Thank you for your comment. The result part in the abstract section was re-written in accordance and highlighted.

5. Follow the format background/intro and aims > methods > results (or mixed methods/results) > conclusions and significance.

Thank you for your comment. The abstract section was corrected in accordance.

6. Graphical abstract – Good basis for graphical abstract, the bottom section however could be adjusted to help the reader understand what they are looking at or how it should be read/interpreted and make a link between the top section (ASE) and it’s use in cells as that’s not clear in the Figure.

Thank you for your suggestion. The bottom section of the graphical abstract was modified in accordance with the comment and interpreted by linking the distinct changes involved in cell death pathways to make it easier to understand for readers.

         Intro

7. Introduction is relatively short but to the point, which is fine.
8. You mention 5-year survival rate has increased overall despite it still being low, but don’t mention the causes for this increase. Is the increase due to work in this field or other general developments in health/medicine? Don’t necessarily need to mention that it has increased as you want to focus on the significance of this work which is that cancer survival is too low and needs improving.

Thank you for your kind notice. The sentence was corrected in accordance.

9. The section on pharmacological drugs is rather broad and doesn’t seem cancer-specific, should add in some further examples.

Thank you for your kind suggestion. The paragraph was re-written in accordance.

10. Careful of punctuation throughout.

Thank you for the suggestion. We have searched them through the whole article and improved the wording.

11. Last ‘paragraph’ of the introduction needs to be broken up into sentences. I also don’t mind using a results-driven descriptive section at the end of the intro, but it should at least be supported by a brief description of what the study aimed to do.

Thank you for the suggestion. We have re-written the last paragraph of the result section and improved the writing. An additional section was added supporting brief description about the study.

Methods

12. Materials and methods relatively thorough
13. Avoid repeating abbreviations that have been mentioned in the introduction i.e. NRF2, STAT3 etc.

Thank you for your comment. The correction has been done in accordance with the comment.

14. For antibodies and cell lines, you should consider adding RRIDs, although not essential if not requested by journal.

Thank you for the comment.

15. Page 3 line 114 - subscript on CO₂

Thank you for your kind notice. We have corrected the subscript.

16. Avoid swapping subheadings between title case and sentence case.

Thank you for the comment. We have searched them through the whole article.

17. ‘Cells were trypsinized’ – using trypsin-EDTA? What %? Or Trypsin alternative. Important information given expression of cell surface receptors and molecules will be impacted by trypsin.

Thank you for your kind notice. In our study, we have utilized 0.25%, Trypsin- EDTA. The correction has been done in accordance.

18. TBS buffer abbreviated without full term.

We appreciate for your comment. The full term was added.

Results

19. Overall figures (in figure legends) need to indicate sample size where not showing representative images. In particular, histograms of Western blot data are described as ‘representative histograms’, but still have error bars and are used for statistical analysis, which should not be done on representative samples, only where data has been pooled from independent biological experiments. Therefore I am unsure if the authors truly mean the histograms are ‘representative’ or if they mean the Western blot images are ‘representative’ and histograms are data analysed from independent replicates?

We appreciate for your comment. Indeed, the histograms represents quantified results of three independent replicates of Western blot analysis. All figure legends were rewritten in accordance with the comment.

20. Figure 1 – Figures 1A and 1B x-axis should be µg/mL? Figure 1B “concentration” is misspelled on the x-axis. Should it not be “ASE concentration” on the X-axis in line with the text (not AS)?

Thank you for your kind notice. The mistakes were corrected in accordance to the comment.

21. Authors chose to use the 72 hour IC₅₀ values, but later incubated these concentrations with cells for 24-48 hours. Can authors comment more specifically on why this was chosen or if there were statistical differences between the IC₅₀ for different times?

Thank you for addressing this point. Initially, in the selection of fixed concentrations of ASE for the further mechanism of action, we didn’t account for the criteria of the IC50 value of the extract. However, findings in other studies discovered the possible chemo drug synergizing effect of ASE indicated the highest combination effect could be achieved at 48 h in co-treatment regimens. Thus this fact gave us a favor to choose 48 h further to investigate the mechanism behind the anti-cancer activity of ASE. 

22. Figure 2 –DTX (positive control) abbreviation not defined.

Thank you for the reviewers’ comments. The full term of DTX was added to the Figure 2 result discussion part.

23. Figure 3 – 3A seems unnecessary and doesn’t provide any additional information that isn’t presented in 3B, especially as the gated area does not appear to align with the measured area? 3B Y-axis is cut off on H23 graph. Also as above, sample size should be indicated as these histograms can’t be ‘representative’ as they have error and statistical analysis associated with them. In 3D, H23 control cells appear to have quite a high and variable cell death in the control cells, is there a reason for this?

Thank you for your comment. All mistakes in Figure 3B, Y-axis and figure legend were revised according to the comment and highlighted. Indeed, we would like to leave Figure 3A in its initial form, as this graph is clearly interpret the results and help the readers quickly understand the technique we used to determine the ROS level.

Also, the reviewer pointed to Figure 3D findings of H23 control cells cell death percentage. In Annexin/7-AAD double staining analysis upon ASE exposure time, we harvested all the cells, including adherent and detached cells, to achieve the most accurate results. During the culture of NSCLC cells, we observed that H23 cells compared to A549, even before any treatment, were easier detached from the surface of the culture dish. We thought this factor could be the reasoned explanation that the H23 control group showed a higher cell death percentage rate than that in A549 cells.

 

 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Thank you for addressing the comments and making the suggested changes. Some final comments:

• NRF2 abbreviation now needs full description in abstract
• Some sub-headings in the methods and results are still not consistent with others regarding title case or sentence case. Should be consistent through manuscript i.e. 2.2 differs from 2.3.
• Figure 3B (H23 cells) y axis label is still half cut off.

 

 

Author Response

Dear Editor and Reviewer,

We sincerely appreciate all valuable comments and suggestions of the Reviewers’ concerning our manuscript entitled “applsci-1674264”. We have carefully considered the comments and tried our best to address minor revision accordingly. The main corrections in the paper and our responses to all the comments are as follows:

 

Comments and Suggestions for Authors

Thank you for addressing the comments and making the suggested changes. Some final comments:

• NRF2 abbreviation now needs full description in the abstract

Thank you for your kind notice. We have added a full description of NRF2 in the abstract section and highlighted it.

• Some sub-headings in the methods and results are still not consistent with others regarding title case or sentence case. Should be consistent through manuscript i.e. 2.2 differs from 2.3.

Thank you for addressing this point. We used sentence cases in sub-headings in the method and results sections and highlighted the corresponding modifications.

• Figure 3B (H23 cells) y axis label is still half cut off.

Thank you for your kind notice. We have fixed the graphs.

Author Response File: Author Response.docx