Novel Semisynthetic Betulinic Acid−Triazole Hybrids with In Vitro Antiproliferative Potential

Round 1

Reviewer 1 Report

This study describes research on the in vitro antiproliferative activity of the novel developed 1,2,4-triazole derivatives of Betulinic acid. The antitumor experiments of these compounds and intermediates were performed on three cancer cell lines and healthy cell lines. And simply explore the anti-tumor mechanism of these compounds. The overall framework of the article is complete and the discussions are sufficient. Some experiments are also very interesting. However, there are still many problems that need to be solved. This study could be accepted after the following revisions.

 

1. In result 2.2, the authors screen out the RPMI-7951 malignant melanoma cells for subsequent experiments, however more intuitive data is needed to support this conclusion. It is suggested that the authors also observe nuclear changes in the other three types of cells (HaCaT, A549 and HT-29) by DAPI staining.

 

2. In result 2.5, there is too little discussion of the results. The authors need to supplement the interpretation of indicators and add some discussion about the results.

 

3. In result 2.6, the authors mentioned that compounds 4a and 4b were suitable for mucosal and cutaneous use. Please give some examples of commercially available products or agents in the development of this type of compound for mucosal and cutaneous use for better understanding.

 

4. In figure 4, the nucleus of DAPI staining is not clear enough, in order to better show the effect and make the reader read, please change the picture with more clarity.

 

5. There are many inconsistencies in the format of this manuscript. For example, the notes format in figure 2 is different from the other figures’ format. The font in table 3 is bold while the font in other tables is not. A space is required between the number and the unit, in line 137, should be 48 h. In addition to these inconsistencies and mistakes pointed out, the author should check the manuscript before accepted.

 

6. The discussion content of the study is too much, which is not conducive to reading. It is suggested that the authors adjust part of the discussion to the results section.

Author Response

1. We sincerely thank the reviewer and are grateful for their time and constructive feedback on our manuscript. All changes to the manuscript were made using Microsoft Word's "Track Changes" feature. Here are the detailed point-by-point responses to the reviewer's suggestions.

   In result 2.2, the authors screen out the RPMI-7951 malignant melanoma cells for subsequent experiments, however more intuitive data is needed to support this conclusion. It is suggested that the authors also observe nuclear changes in the other three types of cells (HaCaT, A549 and HT-29) by DAPI staining.

Answer: We thank the reviewer for this suggestion. The effect of compounds 4a and 4b on nuclear morphology in HaCaT, A549 and HT-29 cell lines have been added to ex-figure 4, now figure 5.

2. In result 2.5, there is too little discussion of the results. The authors need to supplement the interpretation of indicators and add some discussion about the results.

Answer: We thank the reviewer for the useful suggestion. We added some discussions in section 2.5 and removed some text fragments from the discussion section and added the here. Please verify in our revised version of the manuscript.

 

3. In result 2.6, the authors mentioned that compounds 4a and 4b were suitable for mucosal and cutaneous use. Please give some examples of commercially available products or agents in the development of this type of compound for mucosal and cutaneous use for better understanding.

Answer: We thank the reviewer for the suggestion. We added the required information in section 2.6

 

4. In figure 4, the nucleus of DAPI staining is not clear enough, in order to better show the effect and make the reader read, please change the picture with more clarity.

Answer: We thank the reviewer for this observation. Figure 4 has been changed accordingly and is now Figure 5 due to the addition of a supplementary figure.

 

5. There are many inconsistencies in the format of this manuscript. For example, the notes format in figure 2 is different from the other figures’ format. The font in table 3 is bold while the font in other tables is not. A space is required between the number and the unit, in line 137, should be 48 h. In addition to these inconsistencies and mistakes pointed out, the author should check the manuscript before accepted.

Answer: We appreciate the reviewer's insightful comments. We thoroughly reread the text and corrected any inconsistencies that were mentioned as well as any others that were discovered.

6. The discussion content of the study is too much, which is not conducive to reading. It is suggested that the authors adjust part of the discussion to the results section.

Answer: We thank the reviewer for the useful observation. We reduced some of the content in the discussion section and moved it to the result section as suggested.

Reviewer 2 Report

The paper presents the synthesis, characterization, cytotoxic effect and mechanism of anticancer activity of two aryl-substituted-1,2,4-triazole derivatives of betulinic acid.

Although the study concerned only two betulinic acid derivatives with aryl substituted-triazole moieties (compounds 4a and 4b), due to their cytotoxic activity (especially against RPMI-7951 cells) and the investigated mechanism of anticancer action, the publication is worth attention.

This manuscript does require some modifications, which I have outlined in the comments below:

How many replicates of cytotoxicity studies have been performed? This information should be included in the description of the methodology and the results should be given taking into account the SD value.

The cytotoxicity of compounds on cancerous and non-cancer cells should be compared with that of an FDA-approved anticancer drug on the same cell lines.

Author Response

We sincerely thank the reviewer and are grateful for their time and constructive feedback on our manuscript. All changes to the manuscript were made using Microsoft Word's "Track Changes" feature. Here are the detailed point-by-point responses to the reviewer's suggestions.

1. How many replicates of cytotoxicity studies have been performed? This information should be included in the description of the methodology and the results should be given taking into account the SD value.

Answer: We thank the reviewer for this pertinent observation. The missing information has been added in text at Materials and methods, subsection 4.4 Cellular viability.

 

2. The cytotoxicity of compounds on cancerous and non-cancer cells should be compared with that of an FDA-approved anticancer drug on the same cell lines.

 

Answer: We thank the reviewer for this suggestion. We used 5-fluorouracil as positive control and the respective IC50 values were added in table 1.

Reviewer 3 Report

The reviewed article „Novel semisynthetic betulinic acid-triazole hybrids with in 2 vitro antiproliferative potential” seems to be very comprehensive, especially in terms of the evaluation of the mechanism of action and the safety of the tested betulinic acid derivatives. The authors used numerous tests, including assessed the cells viability, Immunofluorescence assay, Mitochondrial respiration assessment, HET-CAM assay.

 

However, I have a few minor comments on the manuscript:

1.      Have the authors verified the effect of the solvent of the test compounds (0.5% DMSO) on the cell lines?

2.      When describing the results of the assessment of cell viability, there are no graphs (histograms) of the dose vs. % cell viability that would better show the inhibitory effect of the test compounds.

3.      The discussion seems to be very exhaustive, but it is an extensive summary and elaboration of the previously described results, and there is little reference to the results of other authors, e.g. a comparison of the IC₅₀ doses obtained by them with the IC₅₀ doses of, for example, betulinic acid and derivatives obtained by other authors for melanoma (which in this case was most sensitive to the tested derivatives).

Author Response

We sincerely thank the reviewer and are grateful for their time and constructive feedback on our manuscript. All changes to the manuscript were made using Microsoft Word's "Track Changes" feature. Here are the detailed point-by-point responses to the reviewer's suggestions.

1. Have the authors verified the effect of the solvent of the test compounds (0.5% DMSO) on the cell lines?

Answer: We thank the reviewer for the excelent observation. Based on previous literature as well as our own studies we know that this concentration does not affect cell viability. Furthermore we used as a negative control DMSO (0.5%) stimulated cells since all other prbes contained 0.5% DMSO. We added all this information into the revised version of our manuscript.

 

2. When describing the results of the assessment of cell viability, there are no graphs (histograms) of the dose vs. % cell viability that would better show the inhibitory effect of the test compounds.

Answer: We thank the reviewer for the usefull sugestion. We added the required histograms as the new Figure 4.

 

3. The discussion seems to be very exhaustive, but it is an extensive summary and elaboration of the previously described results, and there is little reference to the results of other authors, e.g. a comparison of the IC50 doses obtained by them with the IC50 doses of, for example, betulinic acid and derivatives obtained by other authors for melanoma (which in this case was most sensitive to the tested derivatives).

Answer: We thank the reviewer for the usefull observation. We made some adjustments to the disscusion section and moved some parts to the results section as a request from another reviewer. We added some IC50 values, but mainly for BA, beacuse there are curently few triazole derivatives of BA and the ones that we mentioned were not tested on melanoma. That is the main reason as we could not give further examples apart from the ones that we already disscused.

Reviewer 4 Report

The study aimed to evaluate the in vitro antiproliferative activity of 1,2,4-triazole derivatives of betulinic acid. The study is well conducted and the results are relevant.  Some points should be clarified. Many details on HET-CAM assay are missing, in particular, the number of replicates.

Abstract

In “as well as the selective cytotoxicity it exhibits towards tumor cells”, we suggest suppressing “it exhibits towards tumor cells”.

Include more detailed description on HET-CAM assay (chicken breed, conditions of maintenance of the embryos, origin of the embryos; method of application). Were the analyses performed only after 5 minutes? Why longer periods were not considered (i.e. 6 h)?  

Include the number of replicates for HET-CAM assay.

In Discussion, include references for “the immortalized human keratinocytes HaCaT cells were established as skin model since they do not alter the keratinocyte function, cellular responses or differentiation capacity”.

What are the hypothesis to the higher cytotoxicity against RPMI-7951 human malignant melanoma in comparison to the other cell lines? Discuss.

In “Supplementary Materials: The following supporting information can be downloaded at:” the period is incomplete.

Author Response

We sincerely thank the reviewer and are grateful for their time and constructive feedback on our manuscript. All changes to the manuscript were made using Microsoft Word's "Track Changes" feature. Here are the detailed point-by-point responses to the reviewer's suggestions.

1. In “as well as the selective cytotoxicity it exhibits towards tumor cells”, we suggest suppressing “it exhibits towards tumor cells”.

Answer:  We thank the reviewer for the suggestion. We made the required changes.

 

2. Include more detailed description on HET-CAM assay (chicken breed, conditions of maintenance of the embryos, origin of the embryos; method of application). Were the analyses performed only after 5 minutes? Why longer periods were not considered (i.e. 6 h)?

Answer: We thank the reviewer for the well pointed remarks. As requested, we included all the required details in the method section. We performed the test during a 5 minute period as it is given in the ICCVAM-Recommended Test Method Protocol : Hen’ s Egg Test – Chorioallantoic Membrane (HET-CAM) Test Method, which we also cited.

 

3. Include the number of replicates for HET-CAM assay.

Answer: All experiments were done in triplicate. We included the information in the methods section

 

4. In Discussion, include references for “the immortalized human keratinocytes HaCaT cells were established as skin model since they do not alter the keratinocyte function, cellular responses or differentiation capacity”.

Answer: We included the reference for the above mentioned phrase

 

5. What are the hypothesis to the higher cytotoxicity against RPMI-7951 human malignant melanoma in comparison to the other cell lines? Discuss.

Answer: We thank the reviewer for the excellent remark. Given the few literature data on BA triazole derivatives, none of which being tested on melanoma,  we tried to hypothesize the increased activity of the compounds on melanoma based on their parent compound BA.

 

6. In “Supplementary Materials: The following supporting information can be downloaded at:” the period is incomplete.

Answer: We can't finish the sentence because we don't know where the supplementary file will be available. This information will be completed after the editorial team has processed the final proof.

Round 2

Reviewer 1 Report

The paper presents research on the in vitro antiproliferative activity of the novel developed 1,2,4-triazole derivatives of Betulinic acid. Two new compounds were designed which showed promising anticancer effects and prospected their commercial value. After a version of the modification, the author solved most of the problems that had existed in the manuscript. The manuscript has basically met the requirements for publication. The results and discussion have implications for other researchers. The overall paper is well articulated and is suitable for publication in its current form. Readers would like to see more related and follow-up research in the future.

Author Response

We appreciate the reviewer's favorable assessment of our work.