Improvement of Docetaxel Efficacy through Simultaneous Blockade of Transcription Factors NF-κB and STAT-3 Using Pentoxifylline and Stattic in Prostate Cancer Cells

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The submitted manuscript describes the results of the original in vitro research on the effectiveness of Docetaxel with simultaneous blockade using Pentoxifylline and Stattic in context of the therapy of prostate cancer. The manuscript is well written, of high quality and can be published in CIMB after several minor revisions.

Line 47, at this point the Authors should mention the cabazitaxel as alternative to DTX, those options should be briefly compared

In the introduction, a figure presenting chemical structures of Stattic, Docetaxel, Pentoxifylline should be introduced

Stattic has a lot of side effects, don’t you think that K1823 or K1836 would be a better choice?

There are statistical method, even in the GraphPad used by the Authors, to test whether in the current combination of Pentoxifylline and Stattic the observed increase of effectiveness can be called a synergy, defined as an interaction or cooperation giving rise to a whole that is greater than the simple sum of its parts. Please check it and comment.

Line 460, why “Oncology” with capital O?

Line 547, at this point the limitations of the current study should be listed, i.e. the use of the solely one cell line and limitation to solely in vitro

 

 

Author Response

Dear Reviewer:

We appreciate the time and effort spent reviewing our manuscript, as well as your insightful comments and valuable suggestions for improvement. Below, you'll find our responses to your comments, highlighted in red as directed by the journal.

Comments 1: The submitted manuscript describes the results of the original in vitro research on the effectiveness of Docetaxel with simultaneous blockade using Pentoxifylline and Stattic in context of the therapy of prostate cancer. The manuscript is well written, of high quality, and can be published in CIMB after several minor revisions.

Comments 2: Line 47, at this point the Authors should mention the cabazitaxel as alternative to DTX, those options should be briefly compared.

Response 2: Dear reviewer, we appreciate your observation. You are certainly right, cabazitaxel is a relevant alternative to DTX, and we consider it important to mention it in this context. In our revised manuscript, we have conducted a brief comparison between cabazitaxel and DTX, continuing on line 49 to provide a more complete overview.

Comments 3: In the introduction, a figure presenting the chemical structures of Stattic, Docetaxel, and Pentoxifylline should be introduced.

Response 3:  Dear reviewer, we value your comments and have made the necessary adjustments. In our revised manuscript, we have included a table that includes the structure and their CID identifier in PubChem for each drug.

Comments 4: Stattic has a lot of side effects, don’t you think that K1823 or K1836 would be a better choice?

Response 4:  We appreciate your comment. We have used Stattic because it has been important to us in allowing us to explore the implications of targeting STAT3 as a molecular target in cancer therapy with very encouraging results. Stattic is also a well-established and potent STAT3 inhibitor, sheltered by extensive literature. The aim of this work is to determine whether simultaneous inhibition of NF-kB and STAT3 yields improved results, and our findings seem to confirm the hypothesis.  There are reports in the literature on these Stattic analogs, K1836 and K1823. It would be interesting to test them in further studies to give continuity to our research on the STAT3 pathway in cancer, as K1836 and K1823 are reported to retain the ability to inhibit the pathway as well as Stattic while being less toxic, which would make them appropriate for protocols closer to clinical circumstances.

Comments 5: There are statistical methods, even in the GraphPad used by the Authors, to test whether in the current combination of Pentoxifylline and Stattic, the observed increase of effectiveness can be called a synergy, defined as an interaction or cooperation giving rise to a whole that is greater than the simple sum of its parts. Please check it and comment.

Response 5:  We appreciate your comment. Taking into account your recommendation, we have analyzed the synergistic effect of the compounds. However, for this, we have used the web application Synergy Finder, a well-established tool that has been used by other researchers. According to the calculations made by this program, everything indicates that our compounds exhibit synergism at the concentrations with which they were worked. We have included supplementary material (Supplementary Figure and Table 1)  that provides more details about this.

Comments 6: Line 460, why “Oncology” with capital O?

Response 6:  Thank you for pointing that out. The capitalization of 'Oncology' was unintentional. We corrected it to 'oncology' in the revised manuscript.

Comments 7: Line 547, at this point the limitations of the current study should be listed, i.e. the use of the solely one cell line and limitation to solely in vitro.

Response 7:  Dear reviewer, thank you for your valuable feedback. We agree that it is important to acknowledge the limitations of the study and have therefore pointed them out in more detail in the revised manuscript.

We appreciate your understanding and are open to any further suggestions you might have to strengthen our manuscript within the limitations we face.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Colleague,

 

I have carefully reviewed the manuscript titled "Improvement of Docetaxel efficacy through simultaneous blockade of transcription factors NF-κB and STAT-3 using Pentoxifylline and Stattic in Prostate Cancer Cells". As a senior medical researcher with years of experience, I would like to provide my assessment and recommendations.

 

The primary objective of this study was to investigate whether the simultaneous inhibition of NF-kB and STAT-3 could increase the efficacy of docetaxel (DTX) treatment in inducing apoptosis in metastatic castration-resistant prostate cancer (CRPC) DU-145 cells. The authors employed a well-designed experimental approach, utilizing flow cytometry, proliferation assays, colony formation assays, and molecular techniques to evaluate the effects of the combination treatment.

 

The study's key strength lies in its scientific rigor and the comprehensive examination of the underlying mechanisms, including the assessment of caspase activity, mitochondrial membrane potential, and cell cycle progression. The authors have also provided valuable insights into the potential of targeting the NF-κB and STAT-3 pathways to enhance the efficacy of docetaxel in CRPC.

 

To further strengthen the study, I would suggest the following:

 

1. Explore the potential synergistic effects of the combination treatment in an in vivo model to validate the findings and assess the clinical relevance.

2. Investigate the long-term effects of the combination therapy on tumor growth and metastasis, as well as potential adverse events.

3. Examine the impact of the combination treatment on the tumor microenvironment, particularly on immune cell function and the regulation of inflammatory cytokines.

 

Regarding the use of English in the manuscript, I have identified several areas that could be improved:

 

1. The phrase "blocking the translocation of the NF-κB factor to the nucleus" could be more precisely stated as "inhibiting the nuclear translocation of NF-κB".

2. The term "repositioning as an antitumor drug" could be replaced with "repurposing as an antineoplastic agent".

3. The phrase "the latter deriving from the fact that" can be simplified to "because".

4. The use of "henceforth" can be replaced with "hereinafter".

5. The phrase "Fewer than 10 cellular passages were utilized to guarantee maximal resemblance to the original tumor cells" can be improved to "Cells were used within 10 passages to maintain similarity to the original tumor cells".

 

In terms of the strengths and weaknesses of the manuscript, the key strengths are the robust experimental design, the comprehensive investigation of the underlying mechanisms, and the potential clinical implications of the findings. The main weakness is the lack of in vivo validation, which is necessary to further support the translational potential of the combination therapy.

 

In conclusion, this is a well-designed and informative study that contributes to the understanding of the potential therapeutic targeting of the NF-κB and STAT-3 pathways in CRPC. I commend the authors for their efforts and encourage them to continue their investigations to further advance the field of prostate cancer treatment.

 

 

Comments on the Quality of English Language

Dear Colleague,

 

I have carefully reviewed the manuscript titled "Improvement of Docetaxel efficacy through simultaneous blockade of transcription factors NF-κB and STAT-3 using Pentoxifylline and Stattic in Prostate Cancer Cells". As a senior medical researcher with years of experience, I would like to provide my assessment and recommendations.

 

The primary objective of this study was to investigate whether the simultaneous inhibition of NF-kB and STAT-3 could increase the efficacy of docetaxel (DTX) treatment in inducing apoptosis in metastatic castration-resistant prostate cancer (CRPC) DU-145 cells. The authors employed a well-designed experimental approach, utilizing flow cytometry, proliferation assays, colony formation assays, and molecular techniques to evaluate the effects of the combination treatment.

 

The study's key strength lies in its scientific rigor and the comprehensive examination of the underlying mechanisms, including the assessment of caspase activity, mitochondrial membrane potential, and cell cycle progression. The authors have also provided valuable insights into the potential of targeting the NF-κB and STAT-3 pathways to enhance the efficacy of docetaxel in CRPC.

 

To further strengthen the study, I would suggest the following:

 

1. Explore the potential synergistic effects of the combination treatment in an in vivo model to validate the findings and assess the clinical relevance.

2. Investigate the long-term effects of the combination therapy on tumor growth and metastasis, as well as potential adverse events.

3. Examine the impact of the combination treatment on the tumor microenvironment, particularly on immune cell function and the regulation of inflammatory cytokines.

 

Regarding the use of English in the manuscript, I have identified several areas that could be improved:

 

1. The phrase "blocking the translocation of the NF-κB factor to the nucleus" could be more precisely stated as "inhibiting the nuclear translocation of NF-κB".

2. The term "repositioning as an antitumor drug" could be replaced with "repurposing as an antineoplastic agent".

3. The phrase "the latter deriving from the fact that" can be simplified to "because".

4. The use of "henceforth" can be replaced with "hereinafter".

5. The phrase "Fewer than 10 cellular passages were utilized to guarantee maximal resemblance to the original tumor cells" can be improved to "Cells were used within 10 passages to maintain similarity to the original tumor cells".

 

In terms of the strengths and weaknesses of the manuscript, the key strengths are the robust experimental design, the comprehensive investigation of the underlying mechanisms, and the potential clinical implications of the findings. The main weakness is the lack of in vivo validation, which is necessary to further support the translational potential of the combination therapy.

 

In conclusion, this is a well-designed and informative study that contributes to the understanding of the potential therapeutic targeting of the NF-κB and STAT-3 pathways in CRPC. I commend the authors for their efforts and encourage them to continue their investigations to further advance the field of prostate cancer treatment.

 

Author Response

Dear Reviewer:

We appreciate the time and effort spent reviewing our manuscript, as well as your insightful comments and valuable suggestions for improvement. Below, you'll find our responses to your comments, highlighted in red as directed by the journal.

Comments 1: I have carefully reviewed the manuscript titled "Improvement of Docetaxel efficacy through simultaneous blockade of transcription factors NF-κB and STAT-3 using Pentoxifylline and Stattic in Prostate Cancer Cells". As a senior medical researcher with years of experience, I would like to provide my assessment and recommendations. The primary objective of this study was to investigate whether the simultaneous inhibition of NF-kB and STAT-3 could increase the efficacy of docetaxel (DTX) treatment in inducing apoptosis in metastatic castration-resistant prostate cancer (CRPC) DU-145 cells. The authors employed a well-designed experimental approach, utilizing flow cytometry, proliferation assays, colony formation assays, and molecular techniques to evaluate the effects of the combination treatment. The study's key strength lies in its scientific rigor and the comprehensive examination of the underlying mechanisms, including the assessment of caspase activity, mitochondrial membrane potential, and cell cycle progression. The authors have also provided valuable insights into the potential of targeting the NF-κB and STAT-3 pathways to enhance the efficacy of docetaxel in CRPC. To further strengthen the study, I would suggest the following:

Comments 2: Explore the potential synergistic effects of the combination treatment in an in vivo model to validate the findings and assess the clinical relevance.

Response 2: Dear reviewer, we thank you very much for your suggestion on this. Indeed, it would be very interesting to analyze the synergistic effect of the compounds in an in vivo model, which we plan to carry out later in a subsequent study.

Comments 3:  Investigate the long-term effects of the combination therapy on tumor growth and metastasis, as well as potential adverse events.

Response 3:  Dear reviewer, we agree that analyzing the above would be highly valuable. However, due to time limitations in responding to this review, we are unable to conduct this analysis in the current study. We will definitely consider exploring this significant question in future research.

Comments 4: Examine the impact of the combination treatment on the tumor microenvironment, particularly on immune cell function and the regulation of inflammatory cytokines.

Response 4:  Dear reviewer, we appreciate your comment and, concerning this, we have previously investigated some related points, finding that, by inhibiting the STAT3 pathway with Stattic, NK cells were able to increase their cytotoxicity (González-Ochoa, S., et al., 2022) and the phenotype change of macrophages from M2 to M1 (Solís-Martínez R., et al., 2018) was favored. On the other hand, Pentoxifylline was shown to significantly decrease the epithelial-mesenchymal transition of advanced cervical cancer cells by reducing the release of TNF-α and TGF-β1 by blocking the NF-κB pathway (Palafox-Mariscal, L., et al., 2023). Therefore, we agree with your point and intend to investigate this impact in further studies since, in this work, we have been mainly dedicated to exploring the potential of these drugs alone and in combination to lead to enhanced apoptosis in advanced prostate cancer cells and to detail the phenomenon molecularly.

Comments 5: Regarding the use of English in the manuscript, I have identified several areas that could be improved:

1. The phrase "blocking the translocation of the NF-κB factor to the nucleus" could be more precisely stated as "inhibiting the nuclear translocation of NF-κB".
2. The term "repositioning as an antitumor drug" could be replaced with "repurposing as an antineoplastic agent".
3. The phrase "the latter deriving from the fact that" can be simplified to "because".
4. The use of "henceforth" can be replaced with "hereinafter".
5. The phrase "Fewer than 10 cellular passages were utilized to guarantee maximal resemblance to the original tumor cells" can be improved to "Cells were used within 10 passages to maintain similarity to the original tumor cells".

Response 5:  Dear reviewer, we appreciate the feedback. We have taken your suggestions into account and have corrected the aforementioned in the revised manuscript.

Comments 6: In terms of the strengths and weaknesses of the manuscript, the key strengths are the robust experimental design, the comprehensive investigation of the underlying mechanisms, and the potential clinical implications of the findings. The main weakness is the lack of in vivo validation, which is necessary to further support the translational potential of the combination therapy.

Response 6:  Dear reviewer, of course we understand your point, also for us it is of special interest to further extend the knowledge that this research could provide. Thanks to the findings of this study, we have the perspective to examine the effect using in vivo models, but as part of further studies.

Comments 7: In conclusion, this is a well-designed and informative study that contributes to the understanding of the potential therapeutic targeting of the NF-κB and STAT-3 pathways in CRPC. I commend the authors for their efforts and encourage them to continue their investigations to further advance the field of prostate cancer treatment.

Response 7: We sincerely appreciate your thoughtful suggestions, dear reviewer. This work, to our knowledge, represents the first approach to investigate the effects of simultaneously inhibiting the NF-κB and STAT3 pathways, using Pentoxifylline and Stattic, on the induction of enhanced apoptosis in docetaxel-treated advanced prostate cancer cells. The favorable results we have obtained provide a preclinical proof of concept, suggesting that targeting NF-κB and STAT3 could be effective strategies for CRPC. Indeed, some of your suggestions align with future directions we have already planned, aiming to gather more evidence to develop more effective therapeutic options for patients with this disease. Furthermore, these findings could lay the groundwork for future clinical applications. We have, therefore, expanded the discussion of the study's limitations and future perspectives in the revised manuscript.

We appreciate your understanding and are open to any further suggestions you might have to strengthen our manuscript within the limitations we face.

Author Response File: Author Response.pdf