The Anti-Apoptotic Effects of Caspase Inhibitors in Propyl Gallate-Treated Lung Cancer Cells Are Related to Changes in Reactive Oxygen Species and Glutathione Levels
Round 1
Reviewer 1 Report
As described by the author, the anti-apoptotic effects of caspase inhibitors in PG-treated lung cancer cells (Calu-6 and A549 ) are related to changes in ROS and GSH levels. The results showed that PG exhibited antiproliferative activity in lung cancer cells and induced their cell death through apoptosis and necrosis. Increasing the depletion of ROS and GSH levels was accompanied with death of cancer cells and confirmed the caspase inhibitors activity.
In this work, the data and results are good performed and fine clarified, in my opinion this manuscript can be accepted for publication in Molecules after major revision.
Repeating the same sentences should be avoided in this manuscript, for instance in abstract and conclusion parts, the author should rephrase the sentences throughout the manuscript.
The author should precisely identify in the introduction part the caspase roles in apoptosis such as caspase 3 and the process for their activation.
The author carried out the apoptosis and cell cycle assays, at least he should support the discussion with an original figure for the flow cytometry cancer cell cycle distribution and apoptotic status
English language should be refined throughout the manuscript.
Kind regards
Author Response
In this work, the data and results are good performed and fine clarified, in my opinion this manuscript can be accepted for publication in Molecules after major revision.
--> Thank you for your valuable comments and positive evaluations.
Repeating the same sentences should be avoided in this manuscript, for instance in abstract and conclusion parts, the author should rephrase the sentences throughout the manuscript.
--> Thank you for your considerate comment. This manuscript was reviewed by an English editing company, but attempts were made to comprehensively describe or rephrase the sentences throughout the manuscript. Your thoughtful comments will be of great help to future paper writing.
The author should precisely identify in the introduction part the caspase roles in apoptosis such as caspase 3 and the process for their activation.
--> Thank you very much for your comment. I described the roles of caspases in apoptosis and the process for their activation in the Introduction section of the new version of manuscript.
“Apoptosis is a cellular response to cytotoxic drugs. The mechanisms of apoptosis generally involve two main signaling pathways: the mitochondrial pathway and the cell death receptor pathway [20]. The caspase family is a family of cysteine aspartate proteases that are known to play a major role in apoptosis [21, 22]. Members of this family are divided into initiator and executioner caspases. Initiator caspases include caspase-2, caspase-8 and caspase-9, and executioner caspases include caspase-3, caspase-6, and caspase-7 [22]. The executioner caspases exist in cells as inactive dimers and are activated by cleavage [22]. The main substrates for initiator caspases are executioner caspases. The initiation of apoptosis in the mitochondrial pathway is induced or accompanied by increasing the levels of pro-apoptotic proteins, including BAX, and decreasing the levels of anti-apoptotic proteins, including Bcl-2 protein, subsequently causing the loss of mitochondrial membrane potential (MMP; ∆Ψm) [23]. The essential event in this mitochondrial pathway is the efflux of cytochrome c from the mitochondria to cytosol, where it successively forms an apoptosome complex with apoptotic protease-activating factor 1 and caspase-9, consequently leading to the activation of a major executioner caspase, caspase-3 [20, 22, 24]. The pathway related to cell death receptors is characterized by the association of cell death ligands to their death receptors, which stimulates the activities of caspase-8 and caspase-3 [22, 25]. The activation of caspase-3 systematically disassembles cells by cleaving crucial proteins, particularly poly (ADP-ribose) polymerase. Therefore, an attractive strategy for cancer therapy is to target manipulation of the apoptotic pathways.”
The author carried out the apoptosis and cell cycle assays, at least he should support the discussion with an original figure for the flow cytometry cancer cell cycle distribution and apoptotic status.
--> Thank you very much for your comment. I agree with your thought that the data of flow cytometer related to cell cycle distribution and apoptosis should be included in the Figure. However, since there are too many other figures in this paper, the original figures for the flow cytometry have been omitted from this paper for the sake of brevity. Instead, I tried to describe detailed methods of cell cycle distribution and apoptosis analysis and added our previous references related to the methods to Material & Methods section.
2. Materials and Methods
2.5. Cell cycle and sub-G1 cell analysis
Cell cycle and sub-G1 distributions in cells were determined using propidium iodide (PI, Ex/Em = 488 nm/617 nm; Sigma-Aldrich Co.) staining, as previously described [33, 34]…..
2.6. Detection of apoptosis
Apoptosis was measured via annexin V-fluorescein isothiocyanate staining (FITC, Ex/Em = 488/519 nm; Life Technologies, Carlsbad, CA), as previously described [32, 34]…..
English language should be refined throughout the manuscript.
--> Thank you very much for your comment. The paper was reviewed by an English editing company, but attempts were made to correct the errors in English throughout the manuscript.
Reviewer 2 Report
The manuscript, "The anti-apoptotic effects of caspase inhibitors in propyl gallate-treated lung cancer cells are related to changes in reactive oxygen species and glutathione levels " by Woo Hyun Park describes the anti-proliferative effect of propyl gallate (PG) in Calu-6 and A549 lung cancer cells, specifically, in this manuscript, the anti-apoptotic effects of various caspase inhibitors on PG-treated cells were evaluated by verifying reactive oxygen species (ROS) levels and glutathione (GSH) levels.
Major revision:
The manuscript is not very original, especially compared with recently published work by the same author (ref 28: Park, W. H., Propyl gallate reduces the growth of lung cancer cells through caspasedependent apoptosis and G1 phase arrest of 528 the cell cycle. Oncology reports 2020, 44, (6), 2783-2791). The results are difficult to explain and comment on. So the manuscript appears as a list of experiments that are well conceived and well performed, one must say, but leave no room for discussion. In fact, the very paragraph “4. Discussion” merely repeats by making a summary, the results already stated in the dedicated paragraph, as well as illustrating, in the first part, the results reported in reference 28.
Minor revision: In general, the description of the results should be revised more accurately.
Line 184: "Although not statistically significant, treat- 184 ment with inhibitors of pan-caspase, caspase-8, or caspase-9 slightly increased the number 185 of Calu-6 live cells treated with PG (Fig. 1A)" If not statistically significant, it cannot be said that there is a slight increase, also because in the next paragraph, results of the same level, line 217:" None of the tested caspase inhibitors influenced the growth reduction of A549 cells induced by PG". In my opinion the same criterion should be kept in the evaluation of the results.
Line 186: ". In contrast, PG increased the number of Calu- 6 dead cells, and all caspase inhibitors reduced the number of dead cells. In particular, the effect of the caspase-9 inhibitor was significant (Fig. 1B)" In my opinion, these results are not in contrast, but in agreement: decreased survival and increased mortality.
Fig. 6C: Where possible try to equalize the scales of the graphs with those in the same figure.
Author Response
--> Thank you for your valuable comments and positive evaluations.
Major revision:
The manuscript is not very original, especially compared with recently published work by the same author (ref 28: Park, W. H., Propyl gallate reduces the growth of lung cancer cells through caspasedependent apoptosis and G1 phase arrest of 528 the cell cycle. Oncology reports 2020, 44, (6), 2783-2791). The results are difficult to explain and comment on. So the manuscript appears as a list of experiments that are well conceived and well performed, one must say, but leave no room for discussion. In fact, the very paragraph “4. Discussion” merely repeats by making a summary, the results already stated in the dedicated paragraph, as well as illustrating, in the first part, the results reported in reference 28.
--> Thank you for your considerate comment. As you said, I recently published a paper that PG inhibited the growth of Calu-6 and A549 lung cancer cell types through caspase-dependent apoptosis and GSH depletion [30]. Therefore, I focused on investigating the anti-apoptotic effects of various caspase inhibitors in PG-treated Calu-6 and A549 cells in relation to changes in ROS and GSH levels in this study. To provide the reasoning or rationale for this study, I state that “Since PG inhibited the growth of Calu-6 and A549 lung cancer cell types through caspase-dependent apoptosis and GSH depletion [30], the present study focused on investigating the anti-apoptotic effects of various caspase inhibitors in PG-treated Calu-6 and A549 cells with regard to changes in ROS and GSH levels.” in the Discussion section. Your thoughtful comments will be of great help to future research and paper writing.
Minor revision: In general, the description of the results should be revised more accurately.
Line 184: "Although not statistically significant, treat- 184 ment with inhibitors of pan-caspase, caspase-8, or caspase-9 slightly increased the number 185 of Calu-6 live cells treated with PG (Fig. 1A)" If not statistically significant, it cannot be said that there is a slight increase, also because in the next paragraph, results of the same level, line 217:" None of the tested caspase inhibitors influenced the growth reduction of A549 cells induced by PG". In my opinion the same criterion should be kept in the evaluation of the results.
--> Thank you for your thoughtful comments. I agree with your opinion. As you suggested, I removed the phrase “Although not statistically significant,” on Line 184. I also changed the phrase “None of the tested caspase inhibitors influenced …” to the phrase “Treatment with caspase-3 inhibitor slightly attenuated …” on Line 217.
Line 186: ". In contrast, PG increased the number of Calu- 6 dead cells, and all caspase inhibitors reduced the number of dead cells. In particular, the effect of the caspase-9 inhibitor was significant (Fig. 1B)" In my opinion, these results are not in contrast, but in agreement: decreased survival and increased mortality.
--> Thank you for your valuable comments. I agree with you. As you said, on Line 186, I changed “In contrast” to “In addition.” Thank you!
Fig. 6C: Where possible try to equalize the scales of the graphs with those in the same figure.
--> Thank you for your good comment. As you suggested, I equalized the scales of the graphs in Figures 6C and 6D in the new version of manuscript.
I tried to correct the errors in English and revised some minor changes in the whole text in the new version of manuscript. In fact, the previous paper was reviewed by an English editing company.
I appreciate Editor and Reviewer for their considerate cooperation.
Round 2
Reviewer 1 Report
The author is actually replied to the comments which we recommended and ignored some. In my opinion this article in the present form can be accepted for publication in Molecules.
Kind regards
Author Response
Thank you for your valuable comments and positive evaluations.
Reviewer 2 Report
minor revision:
it would be appropriate for the author, not only in Figure 6, to standardize the scales of the graphs
Author Response
As you suggested, the new version of Figures standardized the graph scales of Figure 6 as well as other figures that needed correction. Thank you!!
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
