Anti-Cancer Effects of Lactobacillus plantarum L-14 Cell-Free Extract on Human Malignant Melanoma A375 Cells
, Sangkyu Park 1,2, Jeongmin Seo 1,2,* and Sangho Roh 1,*Round 1
Reviewer 1 Report
This manuscript detailed introduction to exploring the role of chemistry properties of substituted thiazinanes and isomeric forms derivatives. This review detail and highlights the synthetic approaches of thiazinane derivatives and their chemical reactivity. This manuscript content is suitable for publication in Molecules.
Author Response
Response to Reviewer 1 comments
Point 1: This manuscript detailed introduction to exploring the role of chemistry properties of substituted thiazinanes and isomeric forms derivatives. This review detail and highlights the synthetic approaches of thiazinane derivatives and their chemical reactivity. This manuscript content is suitable for publication in Molecules.
Response 1: Thanks for the reviewer’s comments.
Reviewer 2 Report
The paper is of great interest.
I have no major concerns.
My only suggestion is to improve conclusions adding some clinical perspective to the molecular findings. For example, are there clinical trials planned? How do you think a clinical trial of early phase should be planned after this preclinical results?
Author Response
Response to Reviewer 2 comments
The paper is of great interest.
I have no major concerns.
Point 1: My only suggestion is to improve conclusions adding some clinical perspective to the molecular findings. For example, are there clinical trials planned? How do you think a clinical trial of early phase should be planned after this preclinical results?
Response 1: The authors appreciate the reviewer’s kind words and insightful comments. We are planning further studies to find out the effect molecule and to determine doses for clinical trials as mentioned in conclusions.
Page 12, Line 316-317
We think the results of this study are valuable for future clinical trials, but do not have plans to conduct clinical trials in our lab. We are hoping that this study will be utilized to develop therapeutic drugs to treat melanoma using lactic acid bacteria.
Reviewer 3 Report
The article “Anti-cancer Effects of Lactobacillus plantarum L-14 Cell-free Extract on Human Malignant Melanoma Cells” is interesting and well-written. I have some comments and questions that I will list below:
- In the Introduction section (rows 35-37), you state that “However, when metastasis has progressed, chemotherapy and radiotherapy as well as surgery should be used either alone or in combination, causing the survival rate to decrease dramatically”. First, the phrase needs English proofreading. Second, chemotherapy is no longer the mainstay therapy in melanoma – targeted therapy and immunotherapy are currently the norm. Please update the information.
- In sections 2.3. and 2.4. you need to specify the concentrations used since you used different concentrations throughout the experiments
- In the Discussions section, you only talk about Dacarbazine as therapy for melanoma. Currently, immunetherapy and targeted therapy are the most used types of treatment and the Discussions section should clearly include this information
- Although the results are interesting, the article does not talk about practical implications. How would the doses of L-14 used translate to doses currently used in clinical practice?
Minor comments
- Introduction, rows 46-47 – “They are accepted as safe in general when they are applied with adequate doses [11].” – please rephrase (English proofreading required)
- Introduction, rows 47-48 – “Studies have shown that LAB and their metabolites modulated pathogen-induced immune response” instead of “Studies have shown that LAB and their metabolites modulate pathogen-induced immune response”
- Introduction, row 49 – “by reducing cytokine production” instead of “by reducing cytokines production”
- Material and method, rows 94-96 – “The L-14 extract treated groups showed the decreased migration compared to the control group in a dose-dependent manner in both A375 (Figure 3A,C).” - you need to finish the sentence
- Subheading 2.5. “Observation of the release of cytochrome c from the mitochondria following the treatment with L-14 extract” instead of “Observation of the release of cytochrome c from the mitochondria following the treatment of the L-14 extract”
- Rows 182-184 – “In the following experiment, the anti-migratory effect on the L-14 extract showed an additional possibility as a drug for melanoma.” – please rephrase
Author Response
Response to Reviewer 3 comments
The article “Anti-cancer Effects of Lactobacillus plantarum L-14 Cell-free Extract on Human Malignant Melanoma Cells” is interesting and well-written. I have some comments and questions that I will list below:
Major comments
Point 1: In the Introduction section (rows 35-37), you state that “However, when metastasis has progressed, chemotherapy and radiotherapy as well as surgery should be used either alone or in combination, causing the survival rate to decrease dramatically”. First, the phrase needs English proofreading. Second, chemotherapy is no longer the mainstay therapy in melanoma – targeted therapy and immunotherapy are currently the norm. Please update the information.
Response 1: The authors appreciate the reviewer’s kind words and insightful comments. We have corrected the sentence and added the information about targeted therapy and immunotherapy into introduction and discussion.
Page 1, Line 36-39
Page 9, Line 169-174
Point 2: In sections 2.3. and 2.4. you need to specify the concentrations used since you used different concentrations throughout the experiments
Response 2: The authors have added the information about concentrations of the L-14 extract into the manuscript.
Page 5, Line 118
Page 6, Line 132
Point 3: In the Discussions section, you only talk about Dacarbazine as therapy for melanoma. Currently, immunetherapy and targeted therapy are the most used types of treatment and the Discussions section should clearly include this information
Response 3: We agree with the reviewer’s comment, and have added the information about targeted therapy and immunotherapy to prevent to mislead the readers.
Page 9, Line 169-174
Point 4: Although the results are interesting, the article does not talk about practical implications. How would the doses of L-14 used translate to doses currently used in clinical practice?
Response 4: The L-14 extract was used at the concentration of 500 mg/kg in this study. According to the guideline from FDA, Human equivalent dose can be determined by multiply the animal dose by 12.3. In case of the L-14 extract, the dose is 6.15 g/kg when the extract is applied to human. The L-14 extract is a complex of various metabolite and intracellular molecules including exopolysaccharides, proteins, and nucleic acid. We are planning further studies to identify the effect molecule to determine doses of the L-14 for clinical practice.
Minor comments
Point 1: Introduction, rows 46-47 – “They are accepted as safe in general when they are applied with adequate doses [11].” – please rephrase (English proofreading required)
Response 1: The authors have corrected this as the reviewer commented.
Page 2, Line 48 : They are generally considered safe if used at adequate doses.
Point 2: Introduction, rows 47-48 – “Studies have shown that LAB and their metabolites modulated pathogen-induced immune response” instead of “Studies have shown that LAB and their metabolites modulate pathogen-induced immune response”
Response 2: The authors have corrected this as the reviewer commented.
Page 2, Line 49 : modulated --> modulate
Point 3: Introduction, row 49 – “by reducing cytokine production” instead of “by reducing cytokines production”
Response 3: The authors have corrected this as the reviewer commented
Page 2, Line 51 : cytokines --> cytokine
Point 4: Material and method, rows 94-96 – “The L-14 extract treated groups showed the decreased migration compared to the control group in a dose-dependent manner in both A375 (Figure 3A,C).” - you need to finish the sentence.
Response 4: The authors have rephrased the sentence.
Page 4, Line 96-98 : It was shown that migration was reduced in the L-14 extract-treated groups compared to the control group in a dose-dependent manner in both A375 (Figure 3A,C).
Point 5: Subheading 2.5. “Observation of the release of cytochrome c from the mitochondria following the treatment with L-14 extract” instead of “Observation of the release of cytochrome c from the mitochondria following the treatment of the L-14 extract”
Response 5: The authors have corrected this as the reviewer commented.
Page 7, Line 147 : of --> with
Point 6: Rows 182-184 – “In the following experiment, the anti-migratory effect on the L-14 extract showed an additional possibility as a drug for melanoma.” – please rephrase
Response 6: The authors have rephrased the sentence.
Page 9, Line 189-190 : In the next experiment, we confirmed that the L-14 extract had anti-migration effect on A375 cells.
Reviewer 4 Report
The manuscript entitled “Anti-cancer Effects of Lactobacillus plantarum L-14 Cell-free Extract on Human Malignant Melanoma Cells” by Jaehyun Park report L-14 extract demonstrated anticancer activity in melanoma cell lines. Cell viability, migration ability, molecular changes of migration- and apoptosis-related genes, and the location of cytochrome c was evaluated in vitro and also in vivo study confirmed the activity of L-14 extract. Generally, this is an interesting study which might be a novel treatment for melanoma in the future. I have some comments for this manuscript.
Major:
- Only A375 cells (A357P, A375 SM) used in current study is the major limitation. At least two melanoma cell lines should be used, otherwise, we can only conclude that L-14 has activity in A375 but not melanoma.
- The authors only mention chemotherapy in melanoma treatment. Actually, targeted therapy and immunotherapy are main treatment for metastatic melanoma nowadays which significantly improve patient’s survivals. However, the authors did not state this in the section of introduction and discussion which might mislead the readers.
- Some results are different in A375P and A375SM. For example, significantly increased BAX-2 in A375SM mRNA but not in A375P. Authors only mentioned IL-8 in the discussion but I don’t consider this is the man reasons for the different response to L-14.
- For WB, cells were treated with L14 for 72 hours which is very long incubation period and much longer than the qRT-PCR experiments. In addition, 72h treatment kill most cells and few cells were alive for WB. I don’t consider 72h is a good time point for WB. Authors should explain this difference and reason.
- Authors did not investigate how L-14 induced apoptosis. p53 dependent or not ?
- Ki67 is a marker for tumor proliferation. In clinical practice, Protein level by IHC is commonly used for Ki67 assessment but not mRNA level. However, in current study, the authors did not evaluate Ki67 in protein level. The authors may perform IHC from in vivo model which support their findings.
Minor
- Line 25: reagent, L-14 is a reagent?
- Why did the authors choice intraperitoneal injection rather than oral feeding?
- The authors mention L14 works through intrinsic apoptosis but extrinsic apoptosis cannot be excluded by their results.
Author Response
Response to Reviewer 4 comments
The manuscript entitled “Anti-cancer Effects of Lactobacillus plantarum L-14 Cell-free Extract on Human Malignant Melanoma Cells” by Jaehyun Park report L-14 extract demonstrated anticancer activity in melanoma cell lines. Cell viability, migration ability, molecular changes of migration- and apoptosis-related genes, and the location of cytochrome c was evaluated in vitro and also in vivo study confirmed the activity of L-14 extract. Generally, this is an interesting study which might be a novel treatment for melanoma in the future. I have some comments for this manuscript.
Major:
Point 1: Only A375 cells (A357P, A375 SM) used in current study is the major limitation. At least two melanoma cell lines should be used, otherwise, we can only conclude that L-14 has activity in A375 but not melanoma.
Response 1: The authors agree with the reviewer’s comment. We have corrected the expression not to mislead the readers, and added a clause into the main text as the reviewer commented.
Page 2, Line 64
Page 9, Line 211
Page 12, Line 317-319
Point 2: The authors only mention chemotherapy in melanoma treatment. Actually, targeted therapy and immunotherapy are main treatment for metastatic melanoma nowadays which significantly improve patient’s survivals. However, the authors did not state this in the section of introduction and discussion which might mislead the readers.
Response 2: The authors appreciate the reviewer’s kind words and insightful comments. We have added the contents about targeted therapy and immune therapy into the main text.
Page 1, Line 37-39
Page 9, Line 169-174
Point 3: Some results are different in A375P and A375SM. For example, significantly increased BAX-2 in A375SM mRNA but not in A375P. Authors only mentioned IL-8 in the discussion but I don’t consider this is the man reasons for the different response to L-14.
Response 3: The authors agree with the reviewer’s comment. The L-14 extract did not increase mRNA expression of BAX, but increased protein expression of BAX. However, the mRNA level is not always accord with protein level [1]. Although mRNA expression was not changed in A375P, we confirmed that the L-14 extract up-regulated protein expression of BAX in A375P.
IL-8 is not the only difference between A375P and A375SM. Different factors which may affect the difference of the result in this study should be also considered. The previous studies showed that CXCR-1, CXCR-2, and integrin αvβ3 are differentially expressed between A375P and A375SM. It was reported that proliferation, migration, and invasion of A375 was down-regulated by inhibition of CXCR-1 and CXCR-2 [2]. In addition, Apoptosis can be stimulated by loss of integrin-mediated attachment to the extracellular matrix via the action of caspases [3]. We have stated the in discussion and have revised the main text as described above.
Page 9, Line 220-226
[1] Neurol Res. 2006 Dec;28(8):787-93. doi: 10.1179/016164106X110364.
[2] Med Clin (Barc). 2019 Jun 7;152(11):425-430. doi: 10.1016/j.medcli.2018.08.006.
[3] Nat Cell Biol. 2004 May;6(5):388-9. doi: 10.1038/ncb0504-388.
Point 4: For WB, cells were treated with L14 for 72 hours which is very long incubation period and much longer than the qRT-PCR experiments. In addition, 72h treatment kill most cells and few cells were alive for WB. I don’t consider 72h is a good time point for WB. Authors should explain this difference and reason.
Response 4: The authors agree with the reviewer’s comment. The incubation duration for the experiments is an important consideration. According to Figure 4 in [4] and Figure 6. C-H in [5], the protein expression of the apoptosis and EMT markers significantly increased or decreased at 48 h or 72 h rather than at 24 h.
[4] Molecules. 2016 Jun 3;21(6):727. doi: 10.3390/molecules21060727.
[5] PLoS One. 2014 May 23;9(5):e98207. doi: 10.1371/journal.pone.0098207. eCollection 2014.
Point 5: Authors did not investigate how L-14 induced apoptosis. p53 dependent or not ?
Response 5: The investigation of how the L-14 induced apoptosis is an important detail as the reviewer mentioned. In this study, we focused on confirming the efficacy and the possibility of the L-14.Thus, we are planning experiments to investigate the mechanism of action.
Point 6: Ki67 is a marker for tumor proliferation. In clinical practice, Protein level by IHC is commonly used for Ki67 assessment but not mRNA level. However, in current study, the authors did not evaluate Ki67 in protein level. The authors may perform IHC from in vivo model which support their findings.
Response 6: The authors agree with the reviewer’s comment. Unfortunately, the laboratory is currently not set up for IHC. Therefore, we focused on the size of tumor in tissues (the extract-treated tumors decreased to about 40% in weight and about 16% in volume compared to the control. Also, we referred to the study that evaluated cell proliferation using mRNA expression level (Figure 4. a in [6]) . In order to find and isolate the effect molecule, experiments to fractionate the L-14 extract are in progress. In further studies, protein level of Ki67 will be confirmed after setting for IHC.
[6] Neurotox Res. 2019 Oct;36(3):503-514. doi: 10.1007/s12640-019-00040-y.
Minor
Point 1: Line 25: reagent, L-14 is a reagent?
Response 1: We checked the abstract. ‘reagent’ is an error. The sentence has been corrected
Page 1, Line 24-26
Point 2: Why did the authors choice intraperitoneal injection rather than oral feeding?
Response 2: The authors attempted the injection of the L-14 extract in several ways such as intraperitoneal, intravenous and subcutaneous injection (data not shown). Among these, intraperitoneal injection of the extract had the minimal side effects, and showed higher anti-A375 effects. Oral administration was not considered since the L-14 was not used as a form of live lactic acid bacteria.
Point 3: The authors mention L14 works through intrinsic apoptosis but extrinsic apoptosis cannot be excluded by their results.
Response 3: The authors agree with the reviewer’s comments. Therefore, we have highlighted the part and added some sentences into the manuscript to accurately deliver the authors’ intention.
Page 9, Line 207-210
Round 2
Reviewer 4 Report
This manuscript has been improved after authors’ revision. Some minor issue should be noticed.
- The authors have specified melanoma cells as A375, but I still strongly suggest change the title to “… melanoma A375 cells”
- LAB in Abstract should show the full name.
- Line208: Melanoma drug resistance --> melanoma drug resistance
Author Response
Response to Reviewer 4 comments
This manuscript has been improved after authors’ revision. Some minor issue should be noticed.
Point 1: The authors have specified melanoma cells as A375, but I still strongly suggest change the title to “… melanoma A375 cells”
Response 1: The authors appreciate the reviewer’s detailed comment. We have corrected the title as the reviewer commented.
Page 1 Line 3 : Anti-cancer Effects of Lactobacillus plantarum L-14 Cell-free Extract on Human Malignant Melanoma A375 Cells
Point 2: LAB in Abstract should show the full name.
Response 2: The authors missed addition of the abbreviation of lactic acid bacteria after the revision of the last sentence in Abstract. “(LAB)” has inserted after the full name was first mentioned.
Page 1 Line 16: a species of lactic acid bacteria à a species of lactic acid bacteria (LAB)
Point 3: Line208: Melanoma drug resistance --> melanoma drug resistance
Response 3: The authors have corrected the word
Page 9 Line 209 : Melanoma --> melanoma
Author Response File: 
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