Effects of Probiotic Saccharomyces boulardii Supernatant on Viability, Nano-Mechanical Properties of Cytoplasmic Membrane and Pro-Inflammatory Gene Expression in Human Gastric Cancer AGS Cells

Round 1

Reviewer 1 Report

The article "Effects of probiotic Saccharomyces boulardii supernatant on viability, nano-mechanical properties of cytoplasmic membrane and pro-inflammatory gene expression in human gastric cancer AGS cells" is in line with research on the search and testing of new alternatives medicines based on natural biological products to treat patients with gastric cancer.

The authors investigate the anticancer properties of the yeast metabolites and supernatant. The authors investigate the anticancer properties of the yeast metabolites and supernatant. This is particularly interesting, since it allows to prevent the possible negative effect of yeast cells such as fungemia caused by yeast in immunocompromised patients.

The authors were able to demonstrate the dose-dependent antitumor activities of SBS on AGS cell line.

Undoubtedly, the article will be useful for specialists in the treatment of various cancer, but also for specialists in the field of selection of novel medicines for it.

Small remarks:

1. Line 62: “   boulradii” Check the name of the yeast strain.

2. Line 84, 216: “different concentrations of SBS”; “SBS treatments were prepared in 200, 400, 800, 1600 and 3200 μg/mL concentrations”. The concentration of which components of the supernatant is mentioned?

3. Line 86: after 24 and 28 h. Obviously 48 hours?

4. Figure 2:..AFM photographs lack a scale bar!

5. Line 140, Fig. 3:

It's known that survivin is multitasking protein that has dual roles in promoting cell proliferation and preventing apoptosis. Survivin is a component of a chromosome passenger protein complex (CLC) which is essential for chromosome alignment and segregation during mitosis and cytokinesis.

How does a decrease in the level of survivin gene expression affect chromosome segregation and cytokinesis in treated with SBS AGS cells?

6. Why was RPMI1640 medium, which was developed for the cultivation of mammalian cells, used for yeast cultivation, and not standard yeast media: YEPD, SC, YNB?

Author Response

Dear Reviewer 1,

Thank you very much for your valuable, practical and useful comments. This manuscript is thoroughly revised according to your comments and revisions as described below:

1. the name is revised in the text.
2. The concentrations are based on the mixture of lyophilized dried SBS in RPMI solution.
3. “h” is revised to “hours” in the text.
4. The scale of AFM figures is 20 µm. This scale bar is added to the figure and the figure is revised.
5. Survivin also plays as important role in regulating cell proliferation of AGS cells and higher expression of this gene strongly correlates with more viability of gastric cancer cells. In this study, we evaluated the viability of AGS cells to show the anti-proliferative effects of SBS against stomach cancer cells and assess the expression of survivin gene to show the antitumor effects of SBS against AGE cell line. According to your recommendation, we added this explanation in the discussion section and suggested the evaluation of chromosome segregation and cytokinesis in cancer cells treated with SBS for the future studies.
6. I have found that YPD broth was used for the activation and biomass production of the lyophilized S. boulardii strain. It is revised in the text.

Author Response File: Author Response.pdf

Reviewer 2 Report

The present article provides a general overview of the MTT assay, however, lacks in-depth details regarding the cell viability testing. The authors are encouraged to elucidate the details of the cell viability testing process as it holds immense significance in determining the survival of cancer cells. Even the use of water can potentially affect the viability of cancer cells, thus highlighting the importance of meticulous experimental details.

  Moreover, there are certain concerns regarding the authors' method of preparing SBS. Although the article suggests that the SBS solution is prepared using RPMI, which theoretically should not interfere with the experimental results, the RPMI used by the authors contains antibiotics and has undergone a process of drying and re-solubilization with RPMI (supplemented with antibiotics again). This raises concerns about the possibility of the SBS containing elevated levels of antibiotics, which could potentially interfere with the experimental results.

  Furthermore, the authors mentioned DMSO as a negative control. However, DMSO appears to be unrelated to the preparation process of SBS. Notably, in the cell viability experiments, the authors did not present the results of DMSO. It is recommended that the authors provide an explanation for the role of DMSO as a negative control and include the results in the experimental data to can validate their findings.

 

1.        The literature cited in lines 48-55 is not consistent with the author's statement. Therefore, additional references should be provided to support the claim.

2.        The information provided in lines 78-80 is insufficient. The names of the cancer cells previously studied should be clearly stated to provide a better understanding for the readers.

3.        Figure 1: Was a solvent control performed in the solvent experiment? The results indicate that a negative control (solvent) was not performed. Therefore, clarification is required.

4.        Lines 206-216 raise questions about the preparation of SBS. The manuscript states that approximately 100 mg of lyophilized S. boulardii was dissolved in 100 mL of RPMI 1640 cell culture medium supplemented with antibiotics, including penicillin (100 μL/mL) and streptomycin (100 μL/mL), and 10% (v/v) fetal bovine serum. The suspension was then centrifuged for 15 min at 7400 rpm, and the resulting supernatant was collected, passed through a sterilized 0.2 µm filter and lyophilized. The filtered supernatant was then diluted with RPMI 1640 supplemented with FBS and antibiotics.

A.    Why was the lyophilized S. boulardii prepared and concentrated twice? The first preparation suggests that the solubility of RPMI on lyophilized probiotic S. boulardii was 1 mg/ml, and the resulting supernatant was lyophilized again.

B.    Since the first prepared supernatant was lyophilized again in RPMI supplemented with antibiotics, the prepared SBS supernatant may contain twice the concentration of antibiotics. Clarification is required.

C.    How was the solution of SBS prepared as 200, 400, 800, 1600 and 3200 μg/mL? The authors should explain the details of the preparation to validate their experimental design.

D.    What is the exact role of DMSO as the negative control in the experiment? It is unclear from the manuscript whether DMSO was involved in the preparation process. Furthermore, although the authors included DMSO as the negative control in AFM and gene expression analysis, it was not included in the cell viability analysis. Clarification is required.

E.    The authors need to provide further details regarding the cell viability assay because inappropriate experimental design may affect cell viability, and even water may harm cells. The authors should explain how much volume of SBS was added to the cancer cells, and whether appropriate solvent controls were used in this assay.

5.        The details of the cell viability assay are not clear. Please provide a detailed explanation of how the anti-cancer substances and solvent controls were added to the cell culture.

6.        In the conclusion section, lines 285-291, the authors stated that "Regarding the anti-proliferative and antitumor activities of SBS against the AGS cell line, it can be considered a prospective therapeutic strategy to treat human stomach cancer disease." However, a compound that shows anti-proliferative and antitumor activities can also display cellular toxicity. Therefore, it is crucial to conduct safety and toxicity tests on important cells to explain the potential of SBS. In fact, inappropriate solvents in in vitro tests can interfere with cell viability.

Author Response

Dear Reviewer 2,

Thank you very much for your prestigious and practical comments. Your comments helped us to improve strongly the quality of our paper. This manuscript is thoroughly revised according to your comments and revisions as described below:

• In this study, we used MTT assay to evaluate the cell viability of the treated cells and then the antiproliferative effects of SBS against AGS cells were approved by evaluation of survivin gene expression which is strongly associated with the viability of cancer cells. Also, the expression of pro-inflammatory gene was evaluated to describe the antitumor activity of this treatment. Also, using DMSO, as a negative control without any antiproliferative effects against cells, in the same volume showed that the antiproliferative effects of SBS was significant in comparison with the negative controls. This explanation is added into the text.
• I have found that YPD broth was used for the activation and biomass production (growing) of the lyophilized S. boulardii strain. It is revised in the text. Supernatant preparation method in this study was also used previously by several researchers such as Fortin et al. (2018).
• DMSO is commonly used as negative control in the same volume because it does not induce any significant cellular anti-proliferative effects. This explanation is added into the materials and methods section in the text.

1. Regarding the negative side-effects of current strategies for treatment of gastric cancer in humans, citation of the paper by Sexton et al. (2020) is added to this paragraph in the text as an additional and related reference.
2. Information of previous cancer cell lines are added into the text.
3. These results were obtained in comparison with the negative control (treated with DMSO without any anti-proliferative effects on AGS cell line). This explanation is added into the text accordingly.
4. YPD broth was used for the activation and biomass production of the lyophilized S. boulardii strain. It is revised in the text.
5. Lyophilized S. boulardii was live organism. After activation and biomass production (incubation overnight), the supernatant of the produced yeast cells was extracted and dried to prepare different concentrations of the SBS treatments.
6. The method is revised as S. boulardii was initially activated and grown in YPD, previously mentioned.
7. Dried SBS was solubilized in standard RPMI cell culture medium with the mentioned concentrations as the SBS treatments. Each well contanined 100 μL treatment and 100 μL standard cell medium culture. For negative control samples, each well contained 100 μL standard cell medium culture and 100 μL DMSO. This protocol is usually used for antiproliferative evaluation of antitumor compound in cell line model by several researchers. This explanation is added into the text to make it more clear.
8. Since we treated the cells with a definite volume of the treatment (100 μL), we treated the negative control with a standard DMSO solution without any significant cellular anti-proliferative effects in AGS cell line in the same volume (100 μL). The reason of using DMSO as the negative control is also added into the text.
9. The volume and concentrations of SBS treatments are added into the text in details.
10. Details of cell viability, treatments and negative control samples are added into the text.
11. It is revised to potential therapeutic strategy according to your comment. Also, the cell toxicity of SBS is suggested to be evaluated in the future studies. This statement is also added into the conclusion section.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

I am satisfied with the author's response.