Exopolysaccharide from Lactiplantibacillus plantarum YT013 and Its Apoptotic Activity on Gastric Cancer AGS Cells

Round 1

Reviewer 1 Report

In the current study, EPS from L. plantarum YT013 was isolated, and different cancer cells were used to evaluate the anti-cancer ability of EPS and reveal its potential mechanism.

Comments:

1. Line 23: “special medical food supplements” and line 388: development of functional food”.

What do you  really want to achieve? Please clarify   2.How did you think that prevention of gastric cancer can be done? (line 385)   3.Point 3.1.: To make the article easier to read, please add some details about:                - the preparation of L. plantarum YT013 EPS                - the phenol sulfuric acid and carbazole sulfate methods                - the BCA kit Line 90: The precipitate was re-dissolved with deionized water. Please add what volume was used?   4. Point 3.3.: - please add in what did you solubilized the EPS lyophilisate - you forgot to write that cisplatin was used as the reference antitumor drug   5. Point 2.2. -lines 202-203: the current study has nothing to do with the antihypertensive and antiulcer properties of LAB EPS. Please delete them. -Fig 1 (b-f): Cisplatin is not represented   6. Figure 5 is not properly positioned in the manuscript   7. Point 3.9: -add please the number/year of the ethical approuval -it’s not appropriate to write:”Acute oral toxicity analysis” or Test design in acute oral toxicity” - is missing the determination of liver, kidney and hematological toxicity performed on blood samples. Please add   8. line 369, you wrote that “the results could justify further clinical trials”. In my opinion other studies are needed before reaching clinical studies   9.More discussions concerning the obtained results in comparison with other studies already published are nedeed. Please complete.

 

10. You wrote that EPS from Lactiplanti-20 bacillus plantarum YT013 can be an antitumor active drug candidate. Then you wrote about a food supplement. A food supplement can’t be an antitumor active drug candidate.  If you want, it can be used as an adjuvant treatment. Please clarify.

 

Minor editing of English language required

Author Response

Dear reviewer 1:

 

Point 1: Line 23: “special medical food supplements” and line 388: development of functional food”. What do you really want to achieve? Please clarify.

Response 1: Thanks to you for constructive comments and suggestions. Our original intention is to discover a natural anti-tumor drug candidate, and we have revised the inappropriate parts.

 

Point 2: How did you think that prevention of gastric cancer can be done? (line 385).

Response 2: Thank you for your kind suggestion. Our main purpose is to introduce an anti-tumor drug candidate, and we have revised the description for errors.

 

Point 3: Point 3.1.: To make the article easier to read, please add some details about:

- the preparation of L. plantarum YT013 EPS.

- the phenol sulfuric acid and carbazole sulfate methods.

- the BCA kit.

Line 90: The precipitate was re-dissolved with deionized water. Please add what volume was used?

Response 3: Thank you for your kind suggestion. We have made supplements according to the Reviewer’s comments, “The total carbohydrates content was measured using the phenol sulfuric acid method, and glucose was used as the standard, measured at a wavelength of 490 nm. The content of uronic acid was determined by carbazole sulfate method, and glucuronic acid was used as the standard, measured at a wavelength of 530 nm. The protein content of the sample was determined by a BCA kit (Coolaber, Beijing, China), and bovine serum albumin was used as the standard, measured at a wavelength of 590 nm. The moisture content of EPS was determined by a moisture analyzer (Youke Instrument Co., Ltd., Shanghai, China). ” (See page 2, line 87-96).

 

Point 4: Point 3.3.: - please add in what did you solubilized the EPS lyophilisate.

- you forgot to write that cisplatin was used as the reference antitumor drug.

Response 4: Thank you for your kind suggestion. In section 2.3, we have supplemented that “EPS was solubilized in RPMI 1640 or DMEM medium, fresh culture medium was used alone as the control and cisplatin (CIS) was used as the reference antitumor drug. ”(See page 3, line 113).

 

Point 5: Point 2.2. -lines 202-203: the current study has nothing to do with the antihypertensive and antiulcer properties of LAB EPS. Please delete them. -Fig 1 (b-f): Cisplatin is not represented.

Response 5: We thank the reviewer’s valuable suggestion. We have deleted the antihypertensive and antiulcer properties of LAB EPS from the manuscript. In addition, we have made corrections in Fig 1 (b-f). (See Fig 1 (b-f) in revision manuscript).

 

Point 6: Figure 5 is not properly positioned in the manuscript.

Response 6: Thanks a lot for the reviewer’s suggestion. We have changed figure 5 to the end of section 2.6 and before the title of section 2.7. (See page 9, line 315).

 

Point 7: Point 3.9: -add please the number/year of the ethical approuval -it’s not appropriate to write:”Acute oral toxicity analysis” or Test design in acute oral toxicity”- is missing the determination of liver, kidney and hematological toxicity performed on blood samples. Please add.

Response 7: We thank the reviewer’s valuable suggestion. All animal experiments were performed according to animal research guideline of Lanzhou University, and animal experiments were approved by the Ethics Committee of Lanzhou University (the date of approval is 12/28/2021). ”Acute oral toxicity analysis” and “Test design in acute oral toxicity” were corrected as “Biosafety evaluation in mice” and “Acute toxicity test”. Moreover, we mainly observed the histopathological changes of major tissues and organs, and related functional indicators will be tested in the next study.

 

Point 8: line 369, you wrote that “the results could justify further clinical trials”. In my opinion other studies are needed before reaching clinical studies.

Response 8: We acknowledge the reviewer’s comments and suggestions very much. We will consider cconducting preclinical research in the follow-up studies.

 

Point 9: More discussions concerning the obtained results in comparison with other studies already published are nedeed. Please complete.

Response 9: We acknowledge the reviewer’s comments and suggestions very much, which are valuable in improving the quality of our manuscript. We have added more discussions in results and discussion section according to the Reviewer’s comments. The revised portions are marked in red in the revision manuscript.

 

Point 10: You wrote that EPS from Lactiplantibacillus plantarum YT013 can be an antitumor active drug candidate. Then you wrote about a food supplement. A food supplement can’t be an antitumor active drug candidate. If you want, it can be used as an adjuvant treatment. Please clarify.

Response 10: We truly appreciate the reviewer’s comments. Our original intention is to discover an anti-tumor drug candidate, and we have deleted the inappropriate parts such as food supplements.

 

Special thanks to you for your valuable comments.

Reviewer 2 Report

The manuscript of Zhang et al. describes the apoptotic activity of the exopolysaccharide from L. plantarum on gastric cancer AGS cells. The authors show that this effect is dose-dependent. The authors also have shown the safety of the EPS utilization, showing that no significant histopathological change was observed in tissues and organs.

 

Major comments:

-       Material and Methods, section 3.2: Should be given more information on each cell line used.

-       Results and Discussion, section 2.1: The “moisture content of EPS determination” is not described in material and methods section.

-       Results and Discussion, section 2.2, lines 219-220: Please review the results observed. For example, at the concentration of 1000 ug/mL the most significant inhibitory effect occurs at the AGS cells, followed by SGC-7901, PANC-1, HCT116 and the less inhibited is HepG2.

-       Figure 2, 4, 7 – Scale bar missing in the majority of the images.

 

Minor comments:

-       Abstract: Please review the first paragraph. Too long.

-       Material and Methods: When is described % concentration should be indicated if is (V/V) or (W/V). Please review.

-       Material and Methods, line 92: The “phenol sulfuric acid method” measures the total carbohydrates in the sample. So to be more precise instead of “EPS concentration was measured…” should be used “total carbohydrates was measured …”.

-       Material and Methods, line 121: Is missing “Hoechst” before “33258”.

-       Results and Discussion, section 2.1: It is missing the units determined –µg/mL.

-       Figure 3: The figures A to C are described in the legend as photographs, however they are graphical representation of the flow cytometry (one parameter histogram plotting).

-       Results and Discussion: reorganize the figures and text of section 2.3 and 2.4. Start with the text of section 2.3, figure 2, then text of section 2.4 and figure 3.

-       Change Figure 5 to the end of section 2.6 and before the title of section 2.7.

Author Response

Dear reviewer 2:

 

Point 1: Material and Methods, section 3.2: Should be given more information on each cell line used.

Response 1: We truly appreciate the reviewer’s comments. We have added that “The human gastric cancer cell line AGS, colorectal cancer cell line HCT116, liver cancer cell line HepG2 and pancreatic cancer cell line PANC-1 was obtained from the American Type Culture Collection (ATCC, LOT: 70012225, 70019042, 70015966, 70018880). The human gastric cancer cell line SGC-7901 and gastric mucosa cell line GES-1 were supplied by the school of pharmacy, Lanzhou University. ” (See page 3, line 98-101).

 

Point 2: Results and Discussion, section 2.1: The “moisture content of EPS determination” is not described in material and methods section.

Response 2: Thanks a lot for the reviewer’s suggestion. We have supplemented moisture content of EPS determination in the material and methods section based on the reviewer's comments.

 

Point 3: Results and Discussion, section 2.2, lines 219-220: Please review the results observed. For example, at the concentration of 1000 ug/mL the most significant inhibitory effect occurs at the AGS cells, followed by SGC-7901, PANC-1, HCT116 and the less inhibited is HepG2.

Response 3: Thanks for your good instruction. We have made corrections according to the Reviewer’s comments. The revised portions are marked in red in the revision manuscript.

 

Point 4: Figure 2, 4, 7 – Scale bar missing in the majority of the images.

Response 4: We sincerely appreciate the reviewer’s comments. We have supplemented scale bar in figure 2, 4, 7 according to the Reviewer’s comments. (See Figure 2, 4, 7 in revision manuscript).

 

Point 5: Abstract: Please review the first paragraph. Too long.

Response 5: Thanks for your good instruction. We have deleted redundant contents from the abstract. And the relevant contents has been revised according to the Reviewer’s comments.

 

Point 6: Material and Methods: When is described % concentration should be indicated if is (V/V) or (W/V). Please review.

Response 6: Special thanks to you for your kind suggestion. We have made revision accordingly based on the Reviewer’s comments. The revised portions are marked in red in the revision manuscript.

 

Point 7: Material and Methods, line 92: The “phenol sulfuric acid method” measures the total carbohydrates in the sample. So to be more precise instead of “EPS concentration was measured…” should be used “total carbohydrates was measured …”.

Response 7: We truly appreciate the reviewer’s comments. We have made corrections according to the Reviewer’s comments. The original was revised as “the total carbohydrates content of EPS was measured ...”. (See page 2, line 89 and page 5, line 199 ).

 

Point 8: Material and Methods, line 121: Is missing “Hoechst” before “33258”.

Response 8: Thanks a lot for the reviewer’s suggestion. It has been accordingly revised. (See page 3, line124 ).

 

Point 9: Results and Discussion, section 2.1: It is missing the units determined –µg/mL. 

Response 9: Thank you for your comments. Under the same conditions, the standard reference was 1 (100%), and the component content in the sample was expressed as a percentage of the standard, so as to indicated the proportion of the components in the sample.

 

Point 10: Figure 3: The figures A to C are described in the legend as photographs, however they are graphical representation of the flow cytometry (one parameter histogram plotting).

Response 10: We appreciate the reviewer’s suggestion. We have modified the figures descriptions based on the reviewer's comments. The original was revised as “The cell cycle of AGS cells without or with EPS (250 and 500 μg/mL) treatment for 48 h was de-tected by flow cytometry.” (See page 7, line 271 ).

 

Point 11: Results and Discussion: reorganize the figures and text of section 2.3 and 2.4. Start with the text of section 2.3, figure 2, then text of section 2.4 and figure 3.

Response 11: Thank you for your kind suggestion. We have made revision accordingly based on the Reviewer’s comments.

 

Point 12: Change Figure 5 to the end of section 2.6 and before the title of section 2.7.

Response 12: Thanks for your suggestion. We have revised the position of figure 5 according to the reviewer's comments.

 

Special thanks to you for your valuable comments.