Gallic Acid Enhances the Anti-Cancer Effect of Temozolomide in Human Glioma Cell Line via Inhibition of Akt and p38-MAPK Pathway

Round 1

Reviewer 1 Report

Other authors previous reported the co-use of GA and anti-cancer drug have more suitable effects comparing to using single anti-cancer drug. Authors described co-use of cisplatin and GA had more effective for pulmonary small cell carcinoma cell line comparing single use of cisplatin.The results of this report is withint our expected range without new knowledge.

In small aeam authors duscussed the difference of mechanism between GA+TMA for BT cell line and GA+ cisplatin for pulmonary carcinoma cell lie. But mecahnism  of TMZ and cisplaticn are different and BT cells have different biological character comparing pulomonary carcinoma cells. BT is not epithelial malignancy. I think more  experiments are needed.

The morphological findings is needed for this report. The morphological difference between GA and GA+TMA are needed.

Author Response

1. Our study focus on anticancer effect of Ga+TMZ on human glioma cell line and evaluate the mechanisms of the synergism. The structure and pharmacological effect of cisplatin and TMZ is differences. The tumor types treated by cisplatin and TMZ are also different in the clinic. In the discussion section, our purpose is only to cite the reference to indicate that both Ga + TMZ  and Ga+ cisplatin have the identical effect on the inhibition of Bcl-2 expression.
2. We have added the morphological findings in our revised manuscript. The morphological difference between GA and GA+TMA are showed in Figure 1B. The untreated cells and TMZ (200 μM) treatment showed undamaged cellular morphology. Ga (100 μM) decreased total cell numbers. Ga (100 μM) plus TMZ (200 μM) induced a numerous of damaged cells, apoptotic bodies, and membrane blebbing development.

Reviewer 2 Report

I

In the present manuscript, authors have shown that Gallic acid enhances the anti-cancer effect of Temozolomide in human glioma cells by targeting AKT and MAPK pathways. The data presented is not consistent throughout.

1. In figure 1, T+GA 100 combined treatment is showing more than 60% cell death while in Figure 3 combined treatment of T+GA 100 is only showing 27.7% apoptosis. Author should explain why there is such a huge difference in cell death in two different assays.
2. In figure 2 authors should provide CI value for different combinations of drugs. From figure 1 the combined effect of drug appears additive, not synergistic.
3. Reference to figures is missing in the result section.
4. In figure 3 authors should label what different colors represent.
5. In figure 4 Bcl2 expression is very low in both untreated and T+GA 100 samples. While treatment of TMZ and GA alone is showing upregulation of Bcl2 which does not support the author's statement in result section 3.4
6. In figure 5 author should clearly label the Y-axis as DCF fluorescence (MFI). Authors should also include the overlay plot of DCFDA flow cytometry data.

 

Author Response

In the present manuscript, authors have shown that Gallic acid enhances the anti-cancer effect of Temozolomide in human glioma cells by targeting AKT and MAPK pathways. The data presented is not consistent throughout.

1. In figure 1, T+GA 100 combined treatment is showing more than 60% cell death while in Figure 3 combined treatment of T+GA 100 is only showing 27.7% apoptosis. Author should explain why there is such a huge difference in cell death in two different assays.

Response: In Figure 1, the 60% cell death indicates total percentage of cell death. In Figure 3, the 27.7% apoptosis specifically means the percentage of subG1 cells indicating the DNA damage. In addition to apoptosis, it may exist other types of cell death in Figure 1. On the other hand, some cells might be induced apoptotic bodies after T+GA 100 treatment. Apoptotic bodies would rule out by flow cytometer and cannot be detected. It would result in combined treatment of T+GA 100 is only showing 27.7% apoptosis.

2. In figure 2 authors should provide CI value for different combinations of drugs. From figure 1 the combined effect of drug appears additive, not synergistic.

Response: In Figure 1, the two drugs are treated alone and in combination with only one concentration, and it is impossible to actually obtain an additive or synergistic effect from the data. In Figure 2, the two drugs were processed with multiple concentrations and in combinations and used by isobologram analysis. Results show the all points appear under the backslash indicating the synergistic effects.

3. Reference to figures is missing in the result section.

Response: We have added reference to figures in the result section.

4. In figure 3 authors should label what different colors represent.

Response: The color in Figure 3 is displayed after analysis by modfit analysis software. The red peak on the left represents the area of G1 phase. The red peak on the right represents the area of G/M phase. The backslash area in the middle represents the area of S phase. The light blue area in the left represents the area of apoptosis.

5. In figure 4 Bcl2 expression is very low in both untreated and T+GA 100 samples. While treatment of TMZ and GA alone is showing upregulation of Bcl2 which does not support the author's statement in result section 3.4

Response: Bcl 2 is an anti-apoptotic protein. Increase Bcl 2 expression will inhibit the apoptosis effect during anti-cancer drugs treatment. In Figure 4, treatment of TMZ and GA alone is showing upregulation of Bcl2 indicating that an ant-apoptotic effect may exist in treatment of TMZ and GA alone. Bcl2 expression is very low T+GA 100 samples. It may indicate that T+GA 100 samples can induce apoptosis by enhancing the inhibition of Bcl2 expression.  

6. In figure 5 author should clearly label the Y-axis as DCF fluorescence (MFI)

Response: According your comment, we revised the Y-axis label as DCF mean fluorescence intensity (MFI) in Figure 5.

7.  Authors should also include the overlay plot of DCFDA flow cytometry data.

Response: We have included the overlay plot of DCFDA flow cytometer data in Figure 5.

Reviewer 3 Report

The present manuscript shows the effect of temozolomide in combination with gallic acid in the glioblastoma cell line U87MG. The topic is interesting and adds knowledge regarding the use of gallic acid (present in many plant sources), in combination with pharmaceutical drugs, can potentiate cancer therapies, targeting multiple signalling pathways, normally deregulated in cancer, namely proliferation and apoptosis. Unfortunately, the authors did not address the topic how gallic acid reaches the blood stream. This is relevant, and the authors must consider it, and bring for a complete discussion of the results.

 

In the reviewer opinion, there are several topics which need to be further addressed:

 

1. In the abstract section:

-  Line 18, write in full meaning of p.o.;

“TMZ can enter the cerebrospinal fluid by p.o.”

2. In the introduction section:

- line 52, eliminate the word on “with fluoxetine on synergistically enhances”

3. In the material and methods section:

- in the western blot section, the authors should consider emending the word scripted in the line 104 to scraped, as the meaning of the word scripted doesn’t apply how the cells are collected from the plates;

“Cells were scripted and lysed in a lysis buffer”

- Authors should consider to include the constitution of lysis buffer used to lysate the cells, the dilutions for the antibodies used for western blots, as well as the corresponding secondary antibodies, since this information is not described further in the manuscript. Adding this information will add more value to the material and methods section, and promote future reproducibility of the results for future readers;

4. In the results section:

- Line 172, the word of should be removed;

“augmentation in the of SubG1 percentage”

- In the line 186, the author should review the meaning of the words underlined in the sentence.

“Bcl-2 and Bax are important regulated proteins in apoptosis”

It’s not very clear if the authors wanted to state that Bcl-2 and Bax are two proteins important for the regulation of apoptosis or if both proteins are being regulated by other proteins but still they are important for the signaling pathways that triggers apoptosis. It is only perceive what the authors wanted to state later in the discussion section, or either the concepts are not well taken.

 

5. In the discussion section:

- Line 283, replace the word co-incubated to “combination” in the sentence “in co-incubated with clinical chemotherapeutic”

6. There are several typing errors that need to be properly correct.

Author Response

The present manuscript shows the effect of temozolomide in combination with gallic acid in the glioblastoma cell line U87MG. The topic is interesting and adds knowledge regarding the use of gallic acid (present in many plant sources), in combination with pharmaceutical drugs, can potentiate cancer therapies, targeting multiple signalling pathways, normally deregulated in cancer, namely proliferation and apoptosis. Unfortunately, the authors did not address the topic how gallic acid reaches the blood stream. This is relevant, and the authors must consider it, and bring for a complete discussion of the results.

 Response: We have addressed the topic how gallic acid reaches the blood stream in discussion section. 

In the reviewer opinion, there are several topics which need to be further addressed: 

1. In the abstract section:

-  Line 18, write in full meaning of p.o.;

“TMZ can enter the cerebrospinal fluid by p.o.”

Response: p.o.: Abbreviation meaning by mouth, orally (from the Latin "per os", by mouth). 

1. In the introduction section:

- line 52, eliminate the word on “with fluoxetine on synergistically enhances”

Response: The word on has been eliminated.

1. In the material and methods section:

- in the western blot section, the authors should consider emending the word scripted in the line 104 to scraped, as the meaning of the word scripted doesn’t apply how the cells are collected from the plates;

“Cells were scripted and lysed in a lysis buffer”

Response: The “scripted” has revised to “scraped”.

- Authors should consider to include the constitution of lysis buffer used to lysate the cells, the dilutions for the antibodies used for western blots, as well as the corresponding secondary antibodies, since this information is not described further in the manuscript. Adding this information will add more value to the material and methods section, and promote future reproducibility of the results for future readers;

Response: The lysis buffer is PRO-PREP protein extraction solution. It is a commercial solution and purchased from iNtRon Biotechnology (Cat No. 17081.1). The dilution for the first antibodies is 5% bovine serum albumin. The dilution for the secondary antibodies is PBS with Tween® 20 (PBST) solution. We have added the information to the material and methods section.

 

1. In the results section:

- Line 172, the word of should be removed;

“augmentation in the of SubG1 percentage”

Response: The word “of” has been removed.

- In the line 186, the author should review the meaning of the words underlined in the sentence.

“Bcl-2 and Bax are important regulated proteins in apoptosis”

It’s not very clear if the authors wanted to state that Bcl-2 and Bax are two proteins important for the regulation of apoptosis or if both proteins are being regulated by other proteins but still they are important for the signaling pathways that triggers apoptosis. It is only perceive what the authors wanted to state later in the discussion section, or either the concepts are not well taken.

 Response: The sentence has revised to “Bcl-2 and Bax are two proteins important for the regulation of apoptosis”

1. In the discussion section:

- Line 283, replace the word co-incubated to “combination” in the sentence “in co-incubated with clinical chemotherapeutic”

Response: The word “co-incubated” has replaced to “combination”

1. There are several typing errors that need to be properly correct.

Response: We have properly correct several typing errors

Author Response File: Author Response.docx