Astragalus Polysaccharides and Metformin May Have Synergistic Effects on the Apoptosis and Ferroptosis of Lung Adenocarcinoma A549 Cells

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

APS is a common component of Astragalus membranaceust and is known to have various effects, including anti-tumor effects. This study compared the anticancer efficacy with metformin using the A549 cell line, a human lung adenocarcinoma cell, and also confirmed the synergistic effect. This study demonstrated the potential of APS and metformin as adjuvant therapy through in vitro experiments, and also described plans to confirm this efficacy in vivo in the future. I think it is worth accepting this form as article in CIMB.

Author Response

Thank you for your time, expertise, and consideration. We have polished the introduction and methods in the manuscript. We look forward to working with you on the next steps toward publication.

Reviewer 2 Report

Comments and Suggestions for Authors

Reviewer’s Comments:

Title: Astragalus Polysaccharides and Metformin May Have Synergistic Effects on the Apoptosis and Ferroptosis of Lung Adenocarcinoma A549 Cells

The authors have studied the effect of Astragalus polysaccharides and anti-diabetic drug metformin individually and in combination on lung Adenocarcinoma A549 Cells by investigating apoptosis and ferroptosis. The methodology seems to be designed well; however, the content of the manuscript needs major revision. The manuscript (MS) may be recommended for publication after addressing following comments:

1.     All the citations e.g. [x] should be before the full stop.

2.     How could the authors use the term ‘Synergistic’ in title and the manuscript when no models to study synergism have been applied?

3.     ‘Introduction’ section is short and shallow with insufficient information about the APS structure and its role in adjuvant therapy. It’s advised to cite the reference for sentence ‘Traditional……long time’ in lines 42-43 and elaborate the sentence ‘The abilities…reported in lines 44-45. There are numerous publications on antitumor activities of APS and its mechanism of action, that warrants systematic citation in the MS.

4.     ‘Materials and methods’ section seems to be written in a hurry. None of the protocols has reference and a line ‘manufacturer’s instructions’ has been added, which does not provide enough clarity to the readers if one tries to replicate the study. Further no information on the APS has been provided. Once can buy commercial polysaccharides with different molecular weight range. Being commercial samples (APS and metformin), it’s suggested to provide their product codes. Line 79: Authors should mention what different time periods were taken.

5.     Result section

5.1.  Figure1: It’s unclear which graphs are a, b, c and d.

5.2.  Section 3.2: As I mentioned above, the term ‘synergistic’ cannot used unless proven by synergistic studies/models.

Comments on the Quality of English Language

   The language (mainly punctuation, capitalization of words, acronyms) needs to be checked throughout the MS. For instance:

1.1.  Lines 2: ‘May Have’ should be ‘may have’.

1.2.  Acronyms such as DCFDA (line 98), DCFH (line 102) and a few other acronyms have nowhere been explained in the text.

Author Response

Comment 1:   All the citations e.g. [x] should be before the full stop.

Response 1: Thank you for pointing out the citation errors. We have checked the citations in the manuscript and fixed the problems.

Comment 2: How could the authors use the term ‘Synergistic’ in title and the manuscript when no models to study synergism have been applied?

Response 2: Thank you for the insightful opinion. We have updated our results and figures with careful examination. The synergistic effect could be observed in cell viability (page 5, Figure 1), mitochondrial depolarization (page 7, Figure 3), and ferroptosis (page 10, Figure 5). The combinations of APS and metformin strengthened the effect more than using only one remedy.

Comment 3: ‘Introduction’ section is short and shallow with insufficient information about the APS structure and its role in adjuvant therapy. It’s advised to cite the reference for sentence ‘Traditional……long time’ in lines 42-43 and elaborate the sentence ‘The abilities…reported in lines 44-45. There are numerous publications on antitumor activities of APS and its mechanism of action, that warrants systematic citation in the MS.

Response 3: Thank you for the suggestion to improve the introduction. We have enhanced the section on APS to provide readers with a better understanding of it. (From page 2, line 47~80, Astragalus membranaceus is ……… patients with advanced non-small cell lung cancer (NSCLC)[3].) 

       Also we improved the citations from line 42~45 as following: Traditional Chinese Medicine (TCM) has been used in Taiwan, China, Japan, South Korea widely for long time[4]. And there are miscellaneous researches on using TCM as adjuvant therapy on treating cancer. The abilities of enhancing efficacy and decreasing side effects during either chemotherapy or radiotherapy were reported[5]. It has also been reported for lung cancer patients, TCM may serve as possible adjunctive treatment[6].

Comment 4:  ‘Materials and methods’ section seems to be written in a hurry. None of the protocols has reference and a line ‘manufacturer’s instructions’ has been added, which does not provide enough clarity to the readers if one tries to replicate the study. Further no information on the APS has been provided. Once can buy commercial polysaccharides with different molecular weight range. Being commercial samples (APS and metformin), it’s suggested to provide their product codes. Line 79: Authors should mention what different time periods were taken.

Response 4: Thank you for the thoughtful suggestion, in the materials and methods, we have put on our previous study as references and added manufacturer’s instructions for the experiment replication.

The product code and information of APS metformin was provided in line 104~108: The Astragalus polysaccharide (APS) was obtained from Carbosynth. Co. Limited, UK (product code: FA41820, from Mongolian Astragalus using low concentration of ethanol for precipitation and gel chromatography for purification, the approximate molecular weight was 301 kDa), while metformin HCl was obtained from Toronto Research Chemicals, CA, USA (product number: TRC-M258815, molecular weight: 165.62 g/mol).

The XTT time periods was edited for specificity in line 116: Cells were treated with the APS, metformin and their combination for 24 or 48 hours incubation period.

Comment 5: Result section

5.1.  Figure1: It’s unclear which graphs are a, b, c and d.

5.2.  Section 3.2: As I mentioned above, the term ‘synergistic’ cannot used unless proven by synergistic studies/models.

Response 5:  Sorry for our omission, we have uploaded the new figure 1 with a~d order.

The synergistic effect could be observed in cell viability (page 5, Figure 1), mitochondrial depolarization (page 7, Figure 3), and ferroptosis (page 10, Figure 5). The combinations of APS and metformin strengthened the effect more than using only one remedy.

Comments 6: 

Comment 6: 1.1.  Lines 2: ‘May Have’ should be ‘may have’.

1.2.  Acronyms such as DCFDA (line 98), DCFH (line 102) and a few other acronyms have nowhere been explained in the text.

Response 6:  Thank you for pointing out the errors. Line 2 has been edited. And the acronyms were explained when it was first mentioned in the text.

Reviewer 3 Report

Comments and Suggestions for Authors

Opinion

 

on the manuscript by I-Yun Lee et al., entitled "Astragalus Polysaccharides and Metformin May Have Synergistic Effects on the Apoptosis and Ferroptosis of Lung Adenocarcinoma A549 Cells"

 

Manuscript ID: cimb-3040760

 

The manuscript of I-Yun Lee et al. deals with the anticancer action of a polysaccharide mixture of plant origin using lung adenocarcinoma cells. According to the main conclusion of the work, Astragalus polysaccharides (APS) synergize with metformin to decrease the viability of cancer cells. The topic's relevance is outstanding, and the Authors utilized generally accepted cell-based methods. However, its current version contains some crucial insufficiencies which hinder its publication. These are the following points:

 

Major points:

1. Some of the results are not evaluated statistically (e.g., protein expression (Figure 4), DCF fluorescence (panel A of Figure 5)). No robust conclusion can be obtained without statistical evaluation.
2. The reason for metformin's investigation is missing from the introduction. Some hints for previous results would be helpful.
3. The results of the viability assays and their interpretation are not convincing. The combinations are not compared to the single treatments, and therefore, the results presented do not support synergism.
4. The applied APS concentration is a crucial point. Considerable actions were obtained at 10 and 20 mg/ml. Are these concentrations maintainable in an in vivo system? These are extremely high concentrations, so their practical relevance is questionable.
5. According to panel C of Figure 4, APS and the combination decrease the apoptosis rate compared to untreated control. After a more prolonged incubation (panel D) 10 mg/ml APS decreased the apoptotic rate while 20 mg/ml increased it. Is there any explanation for these inconsequent results?
6. Mitochondrial depolarization (Figure 5) was investigated with metformin 10 mM and metformin 5 mM + APS. An appropriate comparison requires the same metformin concentration in combined and single treatments.
7. The origin of APS is missing. Was it extracted by the Authors or purchased?

 

Minor points:

1. Some blots or explanations of Figure 4 have been cut on the right edge.
2. The style of the cited references is not consequent. Some journals are abbreviated (e.g., refs. 3–5, 8), while full names are given for others (e.g., 6, 14–16). Careful checking is suggested.
3. Some of the cited references are defined with authors instead of consecutive numbers: "DeBerardinis & Chandel, 2016; Vander Heiden, Cantley, & Thompson, 2009" (lines 238-239), "Granja et al., 2015" (line 241) and "Zheng et al, 2020" (line 245). Some of these are missing from the list of references.

 

 

Based on all these objections, the rejection of the paper is suggested. 

Author Response

Comment 1: Some of the results are not evaluated statistically (e.g., protein expression (Figure 4), DCF fluorescence (panel A of Figure 5)). No robust conclusion can be obtained without statistical evaluation.

Response 1: Thank you for  pointing out our omission. We have updated the figures in the manuscript. (Figure 4 on page 8, figure 5 on page 10)

Comment 2: The reason for metformin's investigation is missing from the introduction. Some hints for previous results would be helpful.

Response 2: Thank you for the suggestion. To clarify the motivation, we have updated metformin role in cancer treatment and its previous researches in our introduction (Line 79~88, Metformin is the most widely prescribed therapeutic agents for type 2 diabetes… the effect in clinical trials remains inconsistent)

Comment 3: The results of the viability assays and their interpretation are not convincing. The combinations are not compared to the single treatments, and therefore, the results presented do not support synergism.

Response 3: We have improved the interpretation of our results and updated the figures as suggested. In the viability assays, The combinations of APS and metformin strengthened the effect more than using only one remedy. Also, synergism could be observed in the mitochondrial depolarization (page 7, Figure 3), and ferroptosis (page 10, Figure 5).

Comment 4: The applied APS concentration is a crucial point. Considerable actions were obtained at 10 and 20 mg/ml. Are these concentrations maintainable in an in vivo system? These are extremely high concentrations, so their practical relevance is questionable.

Response 4: Thank you for the valuable feedback. It is also our concern regarding the high concentration of APS. In the study, we explored potential synergistic effects to reduce the dosage of these two medications. However, we have not yet determined the precise formulation. Additionally, the dosage used in our cell studies does not directly correlate with in vivo applications. Therefore, further animal studies and pharmacological mechanism experiments are necessary to address these issues comprehensively.
Regarding toxicity concerns, we have referenced the commercial product PG2® Lyo. (injection 500 mg, Drug permit license: no. 058837) approved by the Taiwan FDA. According to their website, a dosage of up to 600 mg/kg/day (36 times of human dosage) was administered intravenously to dogs for 90 consecutive days without any observed toxic effects. (https://www.phytohealth.com.tw/en/medicine/detail)

Comment 5: According to panel C of Figure 4, APS and the combination decrease the apoptosis rate compared to untreated control. After a more prolonged incubation (panel D) 10 mg/ml APS decreased the apoptotic rate while 20 mg/ml increased it. Is there any explanation for these inconsequent results?

Response 5: Thank you for your insightful comments and for providing the opportunity to clarify our findings. In this study, the effects of Astragalus Polysaccharides (APS) on A549 cell apoptosis are overall low, our data still demonstrate significant differences when compared to the control group. Though the difference is subtle, but it may indicate that APS has a measurable impact on the apoptotic pathways of A549 cells.

And the disparity between the results observed at 24-hour and 48-hour incubation periods may suggest the underlying mechanism for APS and metformin over different incubation times. Specifically, the prolonged exposure to APS could be triggering secondary pathways or delayed responses that are not apparent at the 24-hour mark. We hypothesize that APS and metformin may have time-dependent effects on cellular pathways, and we will focus on exploring these mechanisms in greater detail.

Comment 6: Mitochondrial depolarization (Figure 5) was investigated with metformin 10 mM and metformin 5 mM + APS. An appropriate comparison requires the same metformin concentration in combined and single treatments.

Response 6: Thank you for pointing out the issue. We have repeated the experiments using the same metformin concentration for both the combined and single treatments. The results indicated that the combination significantly increased mitochondrial depolarization, suggesting a synergistic effect.

Comment 7: The origin of APS is missing. Was it extracted by the Authors or purchased?

Response 7: The product code and information of APS was provided in line 104~108: The Astragalus polysaccharide (APS) was obtained from Carbosynth. Co. Limited, UK (product code: FA41820, from Mongolian Astragalus using low concentration of ethanol for precipitation and gel chromatography for purification, the approximate molecular weight was 301 kDa)

Comment 8: Some blots or explanations of Figure 4 have been cut on the right edge.

Response 8: Sorry for our omission, we have uploaded the new figure 4.

Comment 9: 1. The style of the cited references is not consequent. Some journals are abbreviated (e.g., refs. 3–5, 8), while full names are given for others (e.g., 6, 14–16). Careful checking is suggested.

2. Some of the cited references are defined with authors instead of consecutive numbers: "DeBerardinis & Chandel, 2016; Vander Heiden, Cantley, & Thompson, 2009" (lines 238-239), "Granja et al., 2015" (line 241) and "Zheng et al, 2020" (line 245). Some of these are missing from the list of references.

Response 9: Thank you for pointing out the citation errors. We have checked the citations in the manuscript and fixed the problems.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have revised the manuscript accordingly. The 'A. membranaceus' in line 64-65 should be italicised. 

Based on the current revision, I would recommend the manuscript for publication. 

 

Author Response

Thank you for accepting our revision of the manuscript. We improved the font in line 64-65. Your valuable feedback has greatly improved our work, and we appreciate your time and effort.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript has been substantially improved but still has some weak points. However, some of the previously raised have not yet been appropriately corrected.

There are still data without statistical evaluation. The column charts of Fig 4 contain no asterisks, but the data are interpreted as treatment-related changes (chapter 3.2). The appropriate statistics are inevitable.

 

All results presented in Figures 3 and 5 are compared only to the control group. These results can not support the proposed synergism if the single-treated and combination-treated groups are not compared directly. 

Author Response

Comment: There are still data without statistical evaluation. The column charts of Fig 4 contain no asterisks, but the data are interpreted as treatment-related changes (chapter 3.2). The appropriate statistics are inevitable.
All results presented in Figures 3 and 5 are compared only to the control group. These results can not support the proposed synergism if the single-treated and combination-treated groups are not compared directly.

Response: 
Thank you for your valuable feedback.
We have carefully re-evaluated our results in Figure 3, 4 and 5. And replication of the western blotting experiments was performed to ensure the accuracy of our findings. We sincerely appreciate your time and effort in reviewing our manuscript.