Influence of Sodium New Houttuyfonate as a New EGFR-TK Inhibitor on the Apoptosis and Autophagy of MCF-7 Cells and Its Toxicity to Caenorhabditis elegans
Round 1
Reviewer 1 Report
Manuscript Title: Influence of sodium new Houttuyfonate as a new EGFR-TK in-2 hibitor on the apoptosis and autophagy of MCF-7 cells and the 3 toxicity to Caenorhabditis elegans
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Comments on the current research article investigating the effect of sodium novel Houttuyfonate as a novel EGFR-TK inhibitor on apoptosis and autophagy of MCF-7 cells and Caenorhabditis elegans toxicity are presented below.
Terms such as “in vivo” and “in vitro” in the article should be written in italics.
Latin names of living things are written in italic or other form, different from the general style of the text in which they are written. Therefore, it would be appropriate to express the Latin names of plants vs living things in italics. my example: Houttuynia cordata
Which botanist or taxonomist gave the true species name of the Houttuynia plant samples used in the experiment, that is, the author who identified the plant? Does this plant species have a herbarium registration number? These are important records in plant studies.
The images of figure2 will be legible if they are displayed larger, not smaller.
Only the cell line "MCF-7 cells" is mentioned by the authors under the heading "2.3. Cell Culture", but not about HeLa.
The numbers on the X-axis of the graph of Figure 3 are sparse. it would be appropriate to display these figures more frequently in order to easily follow the concentrations.
Likewise, it seems that more than 250 concentrations were applied to Hela cells, while the same dose was not applied to MCF-7.
Adding a clearer graph of Figure 5. A will be more beneficial for the reader.
It would be appropriate to add a second graph showing the Wound healing analysis results numerically in Figure 7.
In Figure 8. "NF-kBp65" bands are presented as having very similar intensities at both 0 and 250 doses, but there is a double difference in the numerical graph. Isn't this a contradiction?
You say you're looking at gene expression by PCR, but you're presenting western blot results as a finding, isn't that a contradiction?
If the green fluorescent dots in Figure 9 indicate autophagosomes containing LC3, it would be appropriate to show the numerical amount of all these in a separate graph in the samples.
What does the analysis of cervical cancer cell cytotoxicity have to do with this study?
As an important output of EGFR-TK-targeted molecular docking analysis, shouldn't the effect on the related cellular components in biological material be analyzed?
In the discussion section of the article, the authors should show the close fiction of the docking analyzed substance and apoptosis autophagy nematode toxicity and clearly state the target reached in the study.
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Author Response
Dear Reviewer,
We have studied the valuable comments from you carefully, and tried our best to revise the manuscript. Furthermore, this manuscript has undergone extensive English revisions by Prof. Yucai He who serves as a reviewer for several SCI and EI journals. The point to point responds to your comments are listed as following:
Comments 1: Terms such as “in vivo” and “in vitro” in the article should be written in italics. Latin names of living things are written in italic or other form, different from the general style of the text in which they are written. Therefore, it would be appropriate to express the Latin names of plants vs living things in italics. my example: Houttuynia cordata Which botanist or taxonomist gave the true species name of the Houttuynia plant samples used in the experiment, that is, the author who identified the plant? Does this plant species have a herbarium registration number? These are important records in plant studies.
Response: We have changed the name of Chinese herbal medicine, in vivo, and in vitro to italics. In addition, Houttuynia cordata Thunb., as a traditional Chinese herbal medicine with homologous medicinal and edible origins, is included in the Compendium of Materia Medica, Chinese Materia Medica, and the Dictionary of Traditional Chinese Medicine in which there are many introductions to its properties, efficacy and applicable conditions, but there are not any information about botanist of taxonomist. Moreover, Houttuynia cordata Thunb. in our experiments comes from Shanxi Province and identified by Sifeng Li, and its herbarium registration number is XBGH006851. We have added these above contents in revised manuscript (Line 97-98).
Comment 2: The images of figure 2 will be legible if they are displayed larger, not smaller. Only the cell line "MCF-7 cells" is mentioned by the authors under the heading "2.3. Cell Culture", but not about HeLa.
Response: Thanks for your kind suggestion. We have replaced Figure 2. In addition, we added Hela cells in the corresponding part of cell culture and MTT assay (Line 99, 130, and 135).
Comment 3: The numbers on the X-axis of the graph of Figure 3 are sparse. it would be appropriate to display these figures more frequently in order to easily follow the concentrations. Likewise, it seems that more than 250 concentrations were applied to Hela cells, while the same dose was not applied to MCF-7.
Response: We have redrawn the line chart according to your suggestion. Due to the excessive cell death of MCF-7 cells in the 300ug/ml group and in order to be consistent with other experimental concentrations, this data was not presented.
Comment 4: Adding a clearer graph of Figure 5. A will be more beneficial for the reader.
Response: We have replaced the graphs in Figure 5.
Comment 5: It would be appropriate to add a second graph showing the Wound healing analysis results numerically in Figure 7.
Response: We have added a histogram in Figure 7 (Line 352).
Comment 6: "NF-kBp65" bands are presented as having very similar intensities at both 0 and 250 doses, but there is a double difference in the numerical graph. Isn't this a contradiction?
Response: The bands of C and 250 doses seemed to be similar, but the width and density of bands were analyzed by gel imaging analysis software, and there was indeed a significant difference. Moreover, We conducted a supplementary Western Blotting experiment, and its results are consistent with RT-PCR.(Line 391)
Comment 7: You say you're looking at gene expression by PCR, but you're presenting western blot results as a finding, isn't that a contradiction?
Response：We are sorry not to show the results of western blotting in time due to the COVID-19. Now we have finished it and added the results in the revised manuscript （Line 391）.
Comment 8: If the green fluorescent dots in Figure 9 indicate autophagosomes containing LC3, it would be appropriate to show the numerical amount of all these in a separate graph in the samples.
Response：We have added the numerical amount of autophagosomes containing LC3 in a separate graph (Line 371).
Comment 9: What does the analysis of cervical cancer cell cytotoxicity have to do with this study?
Response：We would like to have a preliminary understanding of the broad-spectrum anti-tumor effect of SNH when we obtained Hela cells from others. In fact, we has also been explored and published antitumor effect of SNH on liver cancer cell lines in our previous articles.
Comment 10: As an important output of EGFR-TK-targeted molecular docking analysis, shouldn't the effect on the related cellular components in biological material be analyzed?
Response：In this study, we initially analyzed the possibility of SNH interacting with common apoptotic receptors FasL and TNF receptors through the DS software docking module, but the results were negative. We also screened EGFR with high expression on many tumors and found that they have good structural affinity. Therefore, it is inferred that SNH may exert antitumor roles by binding to EGFR to antagonize its effects. The subsequent experiments mainly focus on anti-tumor effects, apoptosis, autophagy, and its anti-nematode role. Our further systematic work in the next step will revolve around the molecules in downstream signal pathways of EGFR. Thank you very much for your constructive suggestion.
Comment 11: In the discussion section of the article, the authors should show the close fiction of the docking analyzed substance and apoptosis autophagy nematode toxicity and clearly state the target reached in the study.
Response：We have reorganized and improved the content of the discussion section, and conducted a more systematic and close analysis of the experimental results of each section.
Reviewer 2 Report
Manuscript ID: processes-2328137
Title: Influence of sodium new Houttuyfonate as a new EGFR-TK inhibitor on the apoptosis and autophagy of MCF-7 cells and the toxicity to Caenorhabditis elegans
Reviewer’s comments:
The authors aimed to investigate the effects of SNH on the apoptosis, migration, and autophagy of MCF-7 cell line. The topic is interesting, the current manuscript has been reviewed, and the manuscript needs changes before it can be further processed. The results are clearly discussed. However, there are some major problems that need to be solved.
Introduction:
The grammar and spelling should be carefully checked throughout the manuscript by the authors.
Materials and methods:
Moreover, did the author conduct the gene expression experiments according to MIQE? If so, please attach the complete MIQE reports as supplementary material.
Results:
The protein expression of the markers (VEGF, NF-kB, BCL-2, BAX) used in the present manuscript should be evaluated as well.
Discussion:
I have nothing to report in this part.
The grammar should be carefully checked throughout the manuscript by the authors.
Author Response
Dear Reviewer,
We have studied the valuable comments from you carefully, and tried our best to revise the manuscript. Furthermore, this manuscript has undergone extensive English revisions by Prof. Yucai He who serves as a reviewer for several SCI and EI journals. The point to point responds to your comments are listed as following:
Comment 1: In the part of introduction, the grammar and spelling should be carefully checked throughout the manuscript by the authors.
Response：We have checked and revised the grammar and spelling in introduction part.
Comment 2: In the part of materials and methods, did the author conduct the gene expression experiments according to MIQE? If so, please attach the complete MIQE reports as supplementary material.
Response：We are sorry that we only did traditional PCR experiments instead of qPCR, so we didn't have the MIQE reports. However, in the revised paper, we have supplemented the results of western blotting (Line 349).
Comment 3: In the part of results, The protein expression of the markers (VEGF, NF-kB, BCL-2, BAX) used in the present manuscript should be evaluated as well.
Response：We have represented the results of RT-PCR and western blotting in the revised manuscript (Line391).
Comment 4: The grammar should be carefully checked throughout the manuscript by the authors.
Response：Thank you for your kind suggestion. We have carefully checked and revised the grammar errors of the manuscript.
Reviewer 3 Report
Manuscript entitled "Influence of sodium new Houttuyfonate as a new EGFR-TK inhibitor on the apoptosis and autophagy of MCF-7 cells and the toxicity to Caenorhabditis elegans" require some revisions. Please check comment below
Line 38: mention more important factors
· Line 38: “As everyone knows” please use appropriate words
· Line 55: cite reference
· Line 126-138: remove these unnecessary sentences, methodology section should only mention methods
· Line 154: Please cite reference
· Mention chemical manufacture names and equipment model throughout the manuscript
· Line 169: Model name?
· Line 180: reference:
· Line 200: Check grammar. Experiment repeated 3 times
· Line 202: reference? Most of the method used lack reference
· Line 241: What software used?
· Remove table 2. Write content in text format
· Figure 4: please remove red underlines from figure
· Line 361: avoid these words “ as everyone knows, as is known to all”
· Figure 9: Remove underline from figure, check all figures
· Line 409: where is the reference of previous study
· Discussion section need revisions: Please compare your results with published literature, cite references
Line 38: “As everyone knows” please use appropriate words
Line 200: Check grammar. Experiment repeated 3 times
Line 361: avoid these words “ as everyone knows, as is known to all”
Author Response
Dear Reviewer,
We have studied the valuable comments from you carefully, and tried our best to revise the manuscript. Furthermore, this manuscript has undergone extensive English revisions by Prof. Yucai He who serves as a reviewer for several SCI and EI journals. The point to point responds to your comments are listed as following:
Comment 1: Line 38: mention more important factors
Response：We have added more important factors such as age, genetic predisposition, breast density, obesity, in the revised manuscript.(Line34,35)
Comment 2: Line 38: “As everyone knows” please use appropriate words
Response：“As everyone knows” was replaced by “According to a large amount of previous research”. (Line36)
Comment 3: Line 55: cite reference
Response：we have added a reference 17 .( Line 52）
Comment 4: Line 126-138: remove these unnecessary sentences, methodology section should only mention methods
Response：we have deleted these parts including “Physicochemical parameters play an significant role in controlling the biological activity of chemical entities.” and “Hydrophobic interaction is an important non-bond interaction considerably influencing the interaction of drug molecules and receptors [37], and is the most important role between the non-polar region of the drug and the non-polar region of the receptor. Moreover, TPSA [38] is a useful factor in characterizing drug uptake and is closely related with the hydrogen bonding potential of compounds.”（Line112-116）
Comment 5: Line 154: Please cite reference. Mention chemical manufacture names and equipment model throughout the manuscript
Response：We have added all chemical manufacture and equipment model throughout the manuscript.
Comment 6: Line 169: Model name?
Response：It is the name including the manufacture and model. We have written them separately like “ a Microplate Reader (Bio-Rad 550).”
Comment 7: Line 180: reference
Response：We have done this experiment in accordance with the manufacturer's instructions of Annexin V-FITC/PI apoptosis detection kit. The manufacturer of the latter has been mentioned in materials and reagents section.
Comment 8: Line 200: Check grammar. Experiment repeated 3 times.
Response：”Repeat this test for 3 times” has been replaced by “Experiment repeated 3 times.”(Line 168)
Comment 9: Line 202: reference? Most of the method used lack reference
Response: Line 202 is from reference 39 (reference 42 Line 172 in the revised manuscript）. We have added relative references of all methods.
Comment 10: Line 241: What software used?
Response: All data were plotted using Graghpad przsm 7.0 software. (Line215)
Comment 11: Remove table 2. Write content in text format
Response: We have deleted table 2 and replaced by text description. (Line219-221)
Comment 12: Figure 4: please remove red underlines from figure
Response: We have deleted red underlines from figure 4. (Line282)
Comment 13: Line 361: avoid these words “ as everyone knows, as is known to all”
Response: We have replaced “as is known to all”with “As previous study show”. (Line369)
Comment 14: Figure 9: Remove underline from figure, check all figures
Response: We have check and deleted all red underlines from figures.
Comment 15: Line 409: where is the reference of previous study
Response: References from 54 to 60 are about previous study. (Line440-445)
Comment 16: Discussion section need revisions: Please compare your results with published literature, cite references
Response: We have revised the discussion section carefully and added some relative references. We also corrected many mistakes in grammar. (Line438-510)
Reviewer 4 Report
Title: The title is complex and misleading. Should be changed. Just a suggestion - “Influence of……..and the toxicity to……..” – you can replace ‘the’ with ‘its’.
Abstract: There are numerous misspellings, inappropriate sentences and grammatical errors throughout this manuscript, even in the abstract. Authors are strongly suggested to check and correct those. Scientific names should be italicized. The abstract is casually written. Include specific information and results into it. For example, “In vitro, SNH showed obvious cytotoxicity to MCF-7 cells and significantly decreased cell cloning ability, and SNH of different concentrations promoted the apoptosis of MCF-7 cells….”. Replace ‘significantly’ and ‘different concentrations’ with experimental values. Correct the first and last sentences as well.
Introduction: Please delete casual phrases like ‘As everyone knows’ from the manuscript. “Houttuynia cordata Thunb (HCT) belongs to the Saururaceae which grows in a moist and shady environment. Its pharmaceutical properties are universally known among the people in Asia. Many extracts of HCT have been proofed to have notable anti-inflammatory effects”. - Provide references for these statements.
The third paragraph on antiparasitic drugs should be rearranged as the second last paragraph.
Methods: This section should be checked for more grammatical errors. Examples are: “MCF-7 cells were growth in 10 ml….”, “the viability was analysis by MTT assay”, “After 48 h, remove the medium and add fresh medium. Then, the cells continued to be cultured for more 10d.”
2.6. Annexin V-PI Double Staining: “MCF-7 Cells were collected and mixed it with 195 μl binding buffer”. What was the volume and concentration of MCF-7 cells? What was the cell count? 2.7. Hoechst staining: What was the cell count?
Results: Table 2. It is unclear. As ‘+’ signs indicate toxicity grades, how can we interpret those signs for ‘irritant’ and ‘Mutagenic’?
Fig. 2: This figure is not good enough. Can you provide a better version?
3.3 Cytotoxicity assay: It is important to mention which cell line (Hela or MCF-7) was more sensitive to SNH?
3.4: “This showed that SNH markedly reduced soft agar clonogenesis in MCF-7 cells
at all concentrations, which was consistent with its cytotoxicity “. Figure 4 does not say so. Better activity of SNH was found for 200 µg/ml than 250 µg/ml. Authors should write it clearly. Can the authors explain the possible reason behind it?
3.7: Method says, cells were incubated with SNH for 24 hours. But 48 hours are mentioned in the results. Check and correct this anomaly. The magnification is too low to identify the presence of wrinkled cells, vesicles and apoptotic bodies. Either provide bigger pictures or modify the statement. These pictures are not convincing enough and I could not find significant differences among those.
Fig. 8: Expression of NF-kBp65 in control and treated cells does not match with the text: “In addition, SNH significantly decreased the levels of NF-kBp65 and VEGF in MCF-7 cells…..” I can’t find any difference in expression in the gel figure (8A? Authors should mark the sub-figures as 8A and 8B). It should be ‘SNH’, not ‘SH’. In case of mRNA expression (8B?), difference of the expressions of Bcl-2 and NF-kBp65 are similar. But the gel figure (8A) does not support that. For Bcl-2, we can see the differences in expression clearly, but not for NF-kBp65. Correct the text according to Fig. 8A and 8B.
Discussion: Anti-nematode activity of SNH should be discussed well. It is difficult to correlate with the rest of the study.
There are a lot of mistakes in this manuscript. Authors are suggested to take the necessary help to improve the language.
Author Response
Dear Reviewer,
We have studied the valuable comments from you carefully, and tried our best to revise the manuscript. Furthermore, this manuscript has undergone extensive English revisions by Prof. Yucai He who serves as a reviewer for several SCI and EI journals. The point to point responds to your comments are listed as following:
Comment 1: Title: The title is complex and misleading. Should be changed. Just a suggestion - “Influence of……..and the toxicity to……..” – you can replace ‘the’ with ‘its’.
Response: Thank you for your kind suggestion. We have changed the title into “
Influence of sodium new Houttuyfonate as a new EGFR-TK inhibitor on the apoptosis and autophagy of MCF-7 cells and its toxicity to Caenorhabditis elegans”. (Line3)
Comment 2: Abstract: There are numerous misspellings, inappropriate sentences and grammatical errors throughout this manuscript, even in the abstract. Authors are strongly suggested to check and correct those. Scientific names should be italicized. The abstract is casually written. Include specific information and results into it. For example, “In vitro, SNH showed obvious cytotoxicity to MCF-7 cells and significantly decreased cell cloning ability, and SNH of different concentrations promoted the apoptosis of MCF-7 cells….”. Replace ‘significantly’ and ‘different concentrations’ with experimental values. Correct the first and last sentences as well.
Response: We have adjusted the content of abstract according to your constructive suggestions, and carefully checked the full manuscript and to avoid . (Line11-26)
Comment 3: Introduction: Please delete casual phrases like ‘As everyone knows’ from the manuscript. “Houttuynia cordata Thunb (HCT) belongs to the Saururaceae which grows in a moist and shady environment. Its pharmaceutical properties are universally known among the people in Asia. Many extracts of HCT have been proofed to have notable anti-inflammatory effects”. - Provide references for these statements.
The third paragraph on antiparasitic drugs should be rearranged as the second last paragraph.
Response: We have corrected the content of introduction carefully according to your suggestion. In addition, we have added two references and rearranged the paragraph on antiparasite. (Line30-94)
Comment 4: Methods: This section should be checked for more grammatical errors. Examples are: “MCF-7 cells were growth in 10 ml….”, “the viability was analysis by MTT assay”, “After 48 h, remove the medium and add fresh medium. Then, the cells continued to be cultured for more 10d.”
Response: We have strictly checked and carefully revised grammatical errors, and We have paid special attention to and corrected the incorrect sentences you mentioned.
Comment 5 Methods: 2.6. Annexin V-PI Double Staining: “MCF-7 Cells were collected and mixed it with 195 μl binding buffer”. What was the volume and concentration of MCF-7 cells? What was the cell count? 2.7. Hoechst staining: What was the cell count?
Response: We are very sorry not to write in detail and we have added the number of cells in Annexin V-PI Double Staining (Line151) and Hoechst staining (Line158).
Comment 6: Results: Table 2. It is unclear. As ‘+’ signs indicate toxicity grades, how can we interpret those signs for ‘irritant’ and ‘Mutagenic’?
Response: There are records that Houttuynia cordata has certain mutagenic effects, but it has been widely studied and used in anti-inflammatory therapy in clinical practice because of its significant therapeutic effect. Therefore, we are also interested in its anticancer activity and mechanism, and thus conducted this study.
Comment 7: Results: Fig. 2: This figure is not good enough. Can you provide a better version?
Response: We have place another higher definition photos of Figure 2.(Line256)
Comment 8: Results: 3.3 Cytotoxicity assay: It is important to mention which cell line (Hela or MCF-7) was more sensitive to SNH?
Response: We have added “MFC-7 cells seemed to be more sensitive to SNH than Hela cells” in corresponding position. (Line264)
Comment 9: 3.4: “This showed that SNH markedly reduced soft agar clonogenesis in MCF-7 cells at all concentrations, which was consistent with its cytotoxicity ”. Figure 4 does not say so. Better activity of SNH was found for 200 µg/ml than 250 µg/ml. Authors should write it clearly. Can the authors explain the possible reason behind it?
Response: We have added a detailed description of the cloning ability in 3.4. In addition, the reason of no difference in cloning ability of 200 µg/ml than 250 µg/ml behind this phenomenon was that the size of cell clones in 200 μg/ml group is larger than that of 250 μg/ml group. (Line279-280)
Comment 10: 3.7: Method says, cells were incubated with SNH for 24 hours. But 48 hours are mentioned in the results. Check and correct this anomaly. The magnification is too low to identify the presence of wrinkled cells, vesicles and apoptotic bodies. Either provide bigger pictures or modify the statement. These pictures are not convincing enough and I could not find significant differences among those.
Response: We are very sorry about our carelessness. We have checked the entire manuscript and standardized the cell processing time to 48 hours. Moreover, we have modified the statement of Result 3.7.
Comment 11: Fig. 8: Expression of NF-kBp65 in control and treated cells does not match with the text: “In addition, SNH significantly decreased the levels of NF-kBp65 and VEGF in MCF-7 cells…..” I can’t find any difference in expression in the gel figure (8A? Authors should mark the sub-figures as 8A and 8B). It should be ‘SNH’, not ‘SH’. In case of mRNA expression (8B?), difference of the expressions of Bcl-2 and NF-kBp65 are similar. But the gel figure (8A) does not support that. For Bcl-2, we can see the differences in expression clearly, but not for NF-kBp65. Correct the text according to Fig. 8A and 8B.
Response: The bands of NF-kBp65 in 0 and 250 doses seemed to be similar, but the width and density of bands were analyzed by gel imaging analysis software, and there was indeed a significant difference. Moreover, To make the results of RT-PCR more convincing, We have supplemented the WB experiment, and the levels of related proteins are consistent with the results of RT-PCR. (Line392)
Comment 12: Discussion: Anti-nematode activity of SNH should be discussed well. It is difficult to correlate with the rest of the study.
Response: In discussion, we have reorganized the anti-nematode activity of SNH, which is an indirect evidence of its anti-tumor effect in vivo. (Line505-511)
Comment 13: Comments on the Quality of English Language:There are a lot of mistakes in this manuscript. Authors are suggested to take the necessary help to improve the language.
Response: Thank you for your carefully and professional comments. We have checked and corrected the errors strictly and invited a native English speaker to help us revise the article.
Round 2
Reviewer 1 Report
Comments on the current research article investigating the effect of sodium novel Houttuyfonate as a novel EGFR-TK inhibitor on apoptosis and autophagy of MCF-7 cells and its toxicity against Caenorhabditis elegans are presented below.
Although it has been observed that the authors have made changes in line with the suggestions, we, as the Reviewer, are of the opinion that there are still a number of deficiencies that cause us concerns. In this context;
No satisfactory answers were provided by the authors as to what the analysis of cervical cancer cell cytotoxicity relevance to this study.
In the discussion section of the article, the authors should show the close-up of the docking analysis material with MCF-7 cancer cells and apoptosis autophagy nematode toxicity and should clearly state the target achieved in the study.
Author Response
Comments 1: No satisfactory answers were provided by the authors as to what the analysis of cervical cancer cell cytotoxicity relevance to this study.
Response: We are very sorry about an unsatisfactory answer last time. In the MTT experiment, after exploring the effect of SNH on the viability of MCF-7 cells which belongs to adenocarcinoma, we also want to preliminarily understand its broad-spectrum anticancer activity, so we chose cervical cancer Hela cells as a representative of squamous cell carcinoma which is also one of our future research directions.
Comments 2: In the discussion section of the article, the authors should show the close-up of the docking analysis material with MCF-7 cancer cells and apoptosis autophagy nematode toxicity and should clearly state the target achieved in the study.
Response: We have added the corresponding content about the relationship among Docking results, apoptosis, autophagy, nematode toxicity in the revised manuscript (line498-516).
Reviewer 2 Report
The authors have improved their manuscript according to my observations. The manuscript (processes-2328137) can be accepted in the present form.
Author Response
Comments: The authors have improved their manuscript according to my observations. The manuscript (processes-2328137) can be accepted in the present form.
Response: Thank you very much for your constructive comments and agreement to publish our manuscript in Processes.
Reviewer 3 Report
Authors have addressed all comments, the manuscript is improved and acceptable for publication
Author Response
Comments: Authors have addressed all comments, the manuscript is improved and acceptable for publication.
Response: Thank you very much for your constructive comments and agreement to publish our manuscript in Processes.
Reviewer 4 Report
Please provide a clearer description in details about the NO. 11 comments.
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Comment 11: Fig. 8: Expression of NF-kBp65 in control and treated cells does not match with the text: “In addition, SNH significantly decreased the levels of NF-kBp65 and VEGF in MCF-7 cells…..” I can’t find any difference in expression in the gel figure (8A? Authors should mark the sub-figures as 8A and 8B). It should be ‘SNH’, not ‘SH’. In case of mRNA expression (8B?), difference of the expressions of Bcl-2 and NF-kBp65 are similar. But the gel figure (8A) does not support that. For Bcl-2, we can see the differences in expression clearly, but not for NF-kBp65. Correct the text according to Fig. 8A and 8B.
Author Response
Comments: Please provide a clearer description in details about the NO. 11 comments. Fig. 8: Expression of NF-kBp65 in control and treated cells does not match with the text: “In addition, SNH significantly decreased the levels of NF-kBp65 and VEGF in MCF-7 cells…..” I can’t find any difference in expression in the gel figure (8A? Authors should mark the sub-figures as 8A and 8B). It should be ‘SNH’, not ‘SH’. In case of mRNA expression (8B?), difference of the expressions of Bcl-2 and NF-kBp65 are similar. But the gel figure (8A) does not support that. For Bcl-2, we can see the differences in expression clearly, but not for NF-kBp65. Correct the text according to Fig. 8A and 8B.
Response:Thanks for your favorable comments. We have revised the description especially for about NF-kBp65 in Figure 8. We have marked the results of RT-PCR as Figure 8A and the results of western blotting as Figure 8B. Moreover, SH has been replaced by SNH. (Line370-384)
Round 3
Reviewer 1 Report
Manuscript Title: Influence of sodium new Houttuyfonate as a new EGFR-TK inhibitor on the apoptosis and autophagy of MCF-7 cells and its toxicity to Caenorhabditis elegans
Manuscript ID: Processes-2328137-peer-review-v3
Revision R3
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The following is a commentary on the current research article investigating the effect of sodium novel houttuyfonate as a novel EGFR-TK inhibitor on apoptosis and autophagy of MCF-7 cells and its toxicity towards Caenorhabditis elegans.
It was observed that the authors improved the article by making most, if not all, corrections in line with the referee's suggestions.
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