Q1VA, a Synthetic Chalcone, Induces Apoptosis and Decreases Invasion on Primary Culture of Human Glioblastoma

Round 1

Reviewer 1 Report

In this manuscript, the authors investigated the potential anticancer activities of a new series of synthetic chalcones on glioblastoma. The findings indicate that one chalcone, Q1VA, has cytotoxic effects on GBM cells by inducing apoptosis, depolarizing the mitochondrial membrane, and increasing ROS levels. The observation Q1VA inhibited the invasive behavior of GBM cells further supports the potential therapeutic value of Q1VA for GBM. Overall, this manuscript presents a valuable contribution to the field of GBM research and suggests that synthetic chalcones, particularly Q1VA, may have potential as a novel treatment option for GBM. The authors should consider addressing the suggested improvements before publication.

1.     The authors should use more glioblastoma cell lines to confirm the anti-tumor effect of Q1VA in vitro. They should also perform in vivo experiment to confirm the anti-tumor effect of Q1VA in mouse xenograft model.

2.     The authors do not provide information on the mechanism of action of Q1VA, which could help to further understand its potential as a therapeutic agent.

3.     The group legend in Figure 3 needs to be accurately labeled.

4.     In Figure 7A, a scale should be added.

Author Response

1: The authors should use more glioblastoma cell lines to confirm the anti-tumor effect of Q1VA in vitro. They should also perform in vivo experiment to confirm the anti-tumor effect of Q1VA in mouse xenograft model.

A1: Thank you for your suggestion. We plan to test the chalcones in human GBM U87 cells and carry out in vivo tests in nude mice. However, we would like to present these results in a new publication as it would take more time to start these experiments.

2: The authors do not provide information on the mechanism of action of Q1VA, which could help to further understand its potential as a therapeutic agent.

A2: Thank you for your suggestion. We have included a new figure in the conclusion section (Figure 8) that outlines the proposed mechanism of action for the chalcone Q1VA.

3: The group legend in figure 3 needs to be accurately labeled.

A3: Thank you for pointing out the issue with the group legend in Figure 3. We have revised the caption to accurately label the legend.

4: In Figure 7A, a scale should be added.

A4: Thank you for bringing this to our attention. We have added the necessary scale to Figure 7A.

Author Response File: Author Response.pdf

Reviewer 2 Report

Minor required revisions are as follow:

Abstract

Overall, the title and abstract provide a clear overview of the study and its main findings. However, there are a few areas that could be improved:

• The title could be more specific. Instead of simply stating that Q1VA induces antitumoral effects, it could specify that these effects were observed on a human glioblastoma cell line.
• The abstract could benefit from more specific information about the methods used in the study. For example, it would be helpful to know the concentration of Q1VA used and how long the cells were exposed to it. Additionally, it would be useful to know how the levels of ROS and mitochondrial membrane potential were measured.
• The abstract could also provide more information on the implications of the findings. For example, how might the use of Q1VA be applied in clinical settings? What are the next steps in researching this compound as a potential treatment for GBM?

Introduction:

  In line 3, clarify that gliomas are neoplasms originating from glial cells of the CNS.

specify that the WHO classification refers to gliomas.

  In lines 8-9, provide a brief explanation of why surgery and chemotherapy have not been successful in increasing life expectancy. For example you can explain evidence for the existence of label-retaining tumor-initiating cells (TRICs) in glioblastoma, which are thought to be responsible for the resistance to current treatments. The inclusion of this part would add support to the statement regarding the aggressive nature of glioblastoma and its resistance to current treatments (e.g. Evidence for label-retaining tumour-initiating cells in human glioblastoma.)

  In line 15, clarify that the ABC transporters contribute to chemotherapy resistance.

  In lines 24-25, specify the diseases against which chalcones have shown interesting biological activities.

  In line 38, provide a reference for the 14-month average survival time of patients with GBM.

  In line 47, clarify that the BBB limits the distribution of chemotherapy to the brain.

  In line 55, specify the source of the antimicrobial, anti-inflammatory, analgesic, and antileukemic properties of chalcones.

  In line 64, provide a brief explanation of the mitotic spindle and its role in chromosome segregation.

  In the last sentence, clarify that the objective is to discover new adjuvant drugs for the treatment of brain cancer

Material and methods:

• The first section could be better organized. It would be helpful to start with a brief overview of the synthesis process, followed by a description of the specific methods used for each compound (e.g. Q1VA, Q2VA, Q18VA).
• The abbreviation PTSA is used before it is defined. It would be helpful to provide the full name of the reagent and then introduce the abbreviation in parentheses.
• It is not clear why distilled water was used to facilitate the precipitation of the compounds. A brief explanation or reference to a source that describes this technique would be useful.
• The melting point determination is described in detail, but it would be helpful to also provide information on the purity of the compounds. For example, were the melting points consistent with published values for these compounds?
• The information provided on the NMR and HRMS analysis is detailed and specific, which is good. However, it might be helpful to include a brief explanation of what each type of analysis measures and why it is useful for characterizing the compounds.
• One minor suggestion for improvement would be to provide a brief explanation of the MTT assay principle for readers who may be unfamiliar with the technique.

Results

Overall, the results section appears to be well-written and clearly presents the findings of the study. However, there are a few areas where revisions could be made for clarity:

• In 3.1.1, it would be helpful to specify which cell line(s) were used in the initial screening and subsequent testing of chalcones.
• In 3.1.1, it is unclear what the y-axis of Figure 2A represents. Consider adding a label for clarity.
• In 3.1.1, it is unclear why only Q1VA was selected for further assays. Some justification for this decision would be helpful.
• In 3.2, it would be helpful to specify which phase(s) showed significant differences between the control and Q1VA-treated cells in the PI assay.
• In 3.3, it is unclear what "FCCP" refers to in Figure 5. Consider adding a label or explanation.
• In 3.5, it would be helpful to specify which type of invasive behavior was measured (e.g. migration, invasion, or both).

These are minor revisions, and the results section is otherwise well-written and presents the findings clearly.

Discussion

The discussion section provides a thorough summary of the study's findings, linking the observed effects of chalcone treatment with potential mechanisms of action. The authors have done a good job of contextualizing their results within the existing literature and highlighting the potential of chalcones as a treatment for GBM.

It is suggested to support to the discussion of the potential mechanisms of action of the chalcones studied in the paper, by discussing the role of miRs like miR-21 (e.g. MiR-21: A key player in glioblastoma pathogenesis)

One other suggestion for improvement would be to include a more detailed discussion of the limitations of the study. For example, the authors could discuss potential off-target effects of Q1VA or limitations of the assays used to measure cellular viability and invasiveness. Additionally, it would be helpful to discuss potential avenues for future research to build upon the findings presented in this study.

Overall, the discussion section is well-written and effectively communicates the implications of the study's findings.

Author Response

Reviewer 2:

Minor required revisions are as follow:

Abstract. Overall, the title and abstract provide a clear overview of the study and its main findings. However, there are a few areas that could be improved:

1: The title could be more specific. Instead of simply stating that Q1VA induces antitumoral effects, it could specify that these effects were observed on a human glioblastoma cell line.

A1: Thank you for your suggestion. The title has been revised to specify that the antitumoral effects of Q1VA were observed on a primary culture of human glioblastoma.

2: The abstract could benefit from more specific information about the methods used in the study. For example, it would be helpful to know the concentration of Q1VA used and how long the cells were exposed to it. Additionally, it would be useful to know how the levels of ROS and mitochondrial membrane potential were measured.

A2: Thank you for your suggestion. The abstract has been revised to include specific information about the concentration of Q1VA used (10, 50, and 100 μM) and the duration of cell exposure (24 hours). We also added information on how the levels of ROS and mitochondrial membrane potential were measured (lines 22-26).

3: The abstract could also provide more information on the implications of the findings. For example, how might the use of Q1VA be applied in clinical settings? What are the next steps in researching this compound as a potential treatment for GBM?

A3: Thank you for your suggestion. The abstract has been revised to provide information on the potential application of Q1VA in clinical settings, and the next steps in researching this compound as a potential treatment for GBM (lines 27-3).

Introduction:

4: In line 3, clarify that gliomas are neoplasms originating from glial cells of the CNS.

A4: As suggested by the reviewer, we have revised the text and added the information highlighted in the text.

5: Specify that the WHO classification refers to gliomas.

A5: As suggested by the reviewer, we have revised the text and added the specification to the introduction.

6: In lines 8-9, provide a brief explanation of why surgery and chemotherapy have not been successful in increasing life expectancy. For example, you can explain evidence for the existence of label-retaining tumor-initiating cells (TRICs) in glioblastoma, which are thought to be responsible for the resistance to current treatments. The inclusion of this part would add support to the statement regarding the aggressive nature of glioblastoma and its resistance to current treatments (e.g. Evidence for label-retaining tumour-initiating cells in human glioblastoma.)

A6: As suggested by the reviewer, we have revised the text and added this information to the introduction.

7: In line 15, clarify that the ABC transporters contribute to chemotherapy resistance.

A7: As suggested by the reviewer, we have expanded this topic. We have revised the text to emphasize that the ABC transporters correspond to drug efflux pumps that contribute to chemotherapy resistance.

8: In lines 24-25, specify the diseases against which chalcones have shown interesting biological activities.

A8: As suggested by the reviewer, we have revised the text and added the information needed.

9: In line 38, provide a reference for the 14-month average survival time of patients with GBM.

A9: Thank you for seeing this. As suggested by the reviewer, we have added a reference.

10: In line 47, clarify that the BBB limits the distribution of chemotherapy to the brain.

A10: As suggested by the reviewer, we have revised the text and added the information.

11: In line 55, specify the source of the antimicrobial, anti-inflammatory, analgesic, and antileukemic properties of chalcones.  

A11: As suggested by the reviewer, we have revised the text and added the information.

12: In line 64, provide a brief explanation of the mitotic spindle and its role in chromosome segregation.

A12: As suggested by the reviewer, we have revised the text and added the information.

13: In the last sentence, clarify that the objective is to discover new adjuvant drugs for the treatment of brain cancer.

A13: As suggested by the reviewer, we have revised the text and added the information. Specifically, we have mentioned that the objective of the study is to evaluate the effects of synthetic chalcones on GBM cells with the aim of discovering new adjuvant drugs that can improve the efficacy of current treatments and ultimately improve the prognosis of patients with brain cancer.

Material and methods:

14: The first section could be better organized. It would be helpful to start with a brief overview of the synthesis process, followed by a description of the specific methods used for each compound (e.g. Q1VA, Q2VA, Q18VA).

A14: As suggested by the reviewer, we have revised text and added the information.

15: The abbreviation PTSA is used before it is defined. It would be helpful to provide the full name of the reagent and then introduce the abbreviation in parentheses.

A15: As suggested by the reviewer, we have done de corrections.

16: It is not clear why distilled water was used to facilitate the precipitation of the compounds. A brief explanation or reference to a source that describes this technique would be useful.

A16: As suggested by the reviewer, we have revised text and added the information citing new reference.

17: The melting point determination is described in detail, but it would be helpful to also provide information on the purity of the compounds. For example, were the melting points consistent with published values for these compounds?

A17: We apologize for not including this information earlier. We have now added reference to our previous paper, which also include supporting data for the standard protocol used for the synthesis of chalcones in our university. You can access this data here:

https://www.sciencedirect.com/science/article/abs/pii/S0960894X20304613

18: The information provided on the NMR and HRMS analysis is detailed and specific, which is good. However, it might be helpful to include a brief explanation of what each type of analysis measures and why it is useful for characterizing the compounds.

A18: As suggested by the reviewer, we have revised the text and added the information.

19: One minor suggestion for improvement would be to provide a brief explanation of the MTT assay principle for readers who may be unfamiliar with the technique. A19: A20: As suggested by the reviewer, we have revised the text and added the information.

Results. Overall, the results section appears to be well-written and clearly presents the findings of the study. However, there are a few areas where revisions could be made for clarity:

20: In 3.1.1, it would be helpful to specify which cell line(s) were used in the initial screening and subsequent testing of chalcones.

A20: As suggested by the reviewer, we have revised text and added the information.

21: In 3.1.1, it is unclear what the y-axis of Figure 2A represents. Consider adding a label for clarity. 

A21: Thank you, the label was added.

22: In 3.1.1, it is unclear why only Q1VA was selected for further assays. Some justification for this decision would be helpful.

A22: We have revised the text and included additional information based on our previous experiments. Specifically, we have added information (as data not shown) in the manuscript which demonstrate that higher concentrations of Q1VA can result in a significant reduction in the viability of normal cells, as evidenced by our results from primary cultures of murine astrocytes. While we have provided the graph to the reviewer, we have decided not to include it in the final version of the article as we feel it falls outside the scope of this study.

23: In 3.2, it would be helpful to specify which phase(s) showed significant differences between the control and Q1VA-treated cells in the PI assay.

A23: As suggested by the reviewer, we have revised text and added the information.

24: In 3.3, it is unclear what "FCCP" refers to in Figure 5. Consider adding a label or explanation.

A24: Thank you for the suggest, the description was added to Figure 5.

25: In 3.5, it would be helpful to specify which type of invasive behavior was measured (e.g. migration, invasion, or both).

A25: As suggested by the reviewer, we have revised text and added the information.

Discussion. The discussion section provides a thorough summary of the study's findings, linking the observed effects of chalcone treatment with potential mechanisms of action. The authors have done a good job of contextualizing their results within the existing literature and highlighting the potential of chalcones as a treatment for GBM.

26: It is suggested to support to the discussion of the potential mechanisms of action of the chalcones studied in the paper, by discussing the role of miRs like miR-21 (e.g. MiR-21: A key player in glioblastoma pathogenesis)

A26:  We have added the suggested content to the manuscript.

Q27: One other suggestion for improvement would be to include a more detailed discussion of the limitations of the study. For example, the authors could discuss potential off-target effects of Q1VA or limitations of the assays used to measure cellular viability and invasiveness. Additionally, it would be helpful to discuss potential avenues for future research to build upon the findings presented in this study.

A27: Thank you for this suggestion, the information was added to the final version of the manuscript.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

I am satisfied with the authors' response to my comments. The revised manuscript has been improved and is now ready for publication.

Author Response

Dear 

Thank you for reviewing the paper. We have made the necessary revisions and are sending the final version for publication.

Best regards