Oxyresveratrol in Breast Cancer Cells: Synergistic Effect with Chemotherapeutics Doxorubicin or Melphalan on Proliferation, Cell Cycle Arrest, and Cell Death
, Christian Ferreira 1, Aline Gabrielle Alves de Carvalho 2, Jerson Lima Silva 3, Rafael Garrett 2 and Eliane Fialho 1,*Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsOxyresveratrol is known as a promising anti-cancer drug candidate. Here, Alves Passos et al. report new data about potential synergism between OXY and Doxorubicin and Melphalan.
The experiments are well conducted, the manuscript is nice to read, nevertheless I recommend some corrections:
- The presentation of the results in Chapter 3.1 are not ensuring correct interpretation of the results. Indeed, Data are presented in term of % viability reduction on tumor cells (lines 236-344) and in term of % viability on non-tumor cells (lines 245-245). There is therefore a risk for misinterpretation and transparency.
- FIC > 0.5 can not be attributed as synergism (lines 287-290), see definition (line 123)
Minor comments:
- What is it, line 516?
- Space missing line 551
- Many sentences are not well written, see as example lines 247-252. Please improve the all text, you can often simplify.
Comments on the Quality of English LanguageSee my previous comments
Author Response
1: The presentation of the results in Chapter 3.1 are not ensuring correct interpretation of the results. Indeed, Data are presented in term of % viability reduction on tumor cells (lines 236-344) and in term of % viability on non-tumor cells (lines 245-245). There is therefore a risk for misinterpretation and transparency.
Author´s answer: We changed the presentation of the data as a percentage of viability of non-tumor cells, showing the percentage of reduction in viability as shown in tumor cells (lines 247-249).
2- FIC > 0.5 can not be attributed as synergism (lines 287-290), see definition (line 123).
Author´s answer: Really, we cannot attribute synergism in this way. We had used the wrong references, as the protocol has already been changed in our laboratory. Below is the correct version. The following text and the reference were changed in the Materials and Methods and References Sections:
Isobologram Construction
The fractional inhibitory concentration index (FIC) of OXY was calculated using the formula IC50 of OXY in association with IC50 of OXY alone (IC50, 50% inhibitory concentration) in MCF-7 or MDA-MB-231 cells. The same formula was applied to DOX and MEL. The mean FIC was calculated for each combination and then compared to the reference values and reported as very strong synergism (<0.1), strong synergism (0.1-0.3), synergism (0.3-0.7), moderate synergism (0.7-0.85), slight synergism (0.85-0.90), nearly additive (0.90-1.10), slight antagonism (1.10-1.20), moderate antagonism (1.20-1.45), antagonism (1.45-3.3), strong antagonism (3.3-10) and very strong antagonism (>10). The synergistic effect between OXY + DOX and OXY + MEL were analyzed using FIC values to plot the isobologram, according to the method of Chou (2006) [13]. Antagonistic combinations were defined as points above the line of additivity, and synergistic combinations were defined as points below the line.
Reference 13: Chou, T.C. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol. Rev. 2006, 58, 3, 621-81. doi: 10.1124/pr.58.3.10.
Minor comments:
3- What is it, line 516?
Author´s answer: We changed the sentence for: “Our results of the clonogenic assay support our findings in the MTT assay.” (line 554).
4- Space missing line 551.
Author´s answer: We separete the paragraphs into lines 571-584 and 585-597.
5- Many sentences are not well written, see as example lines 247-252. Please improve the all text, you can often simplify.
Author´s answer: The suggestion was accepted and the text was modified.
Before:
We determined the IC50 of OXY, DOX, and MEL for MCF-10A, MCF-7, and MDA-MB-231 cells were ˃300, 26.54, and 195.97 µM, 164.10, 22.60 and 155.70 µM, and 287.08, 32.55 and 240.26 µM, respectively (Table 1). The selectivity index (SI) was also calculated from the IC50 obtained from the MCF-10A cells and the IC50 from the MCF-7 and MDA-MB-231 cells, the SI for OXY was ˃1.83 and ˃1.05, while for DOX and MEL were 1.17 and 1.26, and 0.82 and 0.82, respectively (Table 1).
Modified:
The IC50 of OXY, DOX, and MEL were determined, with values of ˃300, 26.54, and 195.97 µM for MCF-10A, 164.10, 22.60 and 155.70 µM for MCF-7 and 287.08, 32.55 and 240.26 µM for MDA-MB-231 cells, respectively (Table 1). The selectivity index (SI) was also calculated from the IC50 obtained from the MCF-10A cells and the IC50 from the MCF-7 and MDA-MB-231 cells, the SI for OXY was ˃1.83 and ˃1.05, while for DOX were 1.17 and 1.26, and for MEL were 0.82 and 0.82, respectively (Table 1). (lines 250-255).
Reviewer 2 Report
Comments and Suggestions for AuthorsIn this study the authors investigated the cytotoxic effect of Oxyresveratrol, Doxorubicin and Melphalan alone or associated in an in vitro model of breast cancer. The results showed a synergistic effect of Oxyresveratrol with chemotherapeutic agents to alter the cell cycle and led the cells to necrosis or to apoptosis. Overall, the study is informative and provides new insights for further exploration in the field.
I find that the manuscript offers interesting insights on the topic of chemoresistance. Although the results demonstrate a beneficial effect deriving from the use of OXY+DOX or OXY+MEL, we are, unfortunately, very far from offering new strategies for future animal studies and clinical trials. In fact, the data should first be confirmed on different primary and immortalized cell lines and with a broader set of markers (such as PI3K/AKT signaling). Therefore, it would be desirable to review the conclusions. However, I would say that the manuscript lays excellent foundations for future studies.
In my opinion, given the results provided, it could be considered for publication after revision, particularly in terms resolution and size of images. Furthermore, there are some minor writing style mistakes that should be corrected.
What, in my opinion, strongly penalizes the work are the images.
Figure 1 (A–C), it is impossible to read any numerical value. The figures are very small, the characters are microscopic, and the choice of gray scale is not very effective.
Table 2, in my opinion, should be completely formatted
Figure 2 (C-D), it is almost impossible to appreciate the result
Figure 4 (P and H), it is almost impossible to appreciate the result.
Comments on the Quality of English Language
There are some minor writing style mistakes that should be corrected.
Author Response
In this study the authors investigated the cytotoxic effect of Oxyresveratrol, Doxorubicin and Melphalan alone or associated in an in vitro model of breast cancer. The results showed a synergistic effect of Oxyresveratrol with chemotherapeutic agents to alter the cell cycle and led the cells to necrosis or to apoptosis. Overall, the study is informative and provides new insights for further exploration in the field.
I find that the manuscript offers interesting insights on the topic of chemoresistance. Although the results demonstrate a beneficial effect deriving from the use of OXY+DOX or OXY+MEL, we are, unfortunately, very far from offering new strategies for future animal studies and clinical trials. In fact, the data should first be confirmed on different primary and immortalized cell lines and with a broader set of markers (such as PI3K/AKT signaling). Therefore, it would be desirable to review the conclusions. However, I would say that the manuscript lays excellent foundations for future studies.
In my opinion, given the results provided, it could be considered for publication after revision, particularly in terms resolution and size of images. Furthermore, there are some minor writing style mistakes that should be corrected.
What, in my opinion, strongly penalizes the work are the images.
Author´s answer: The referee is correct about resolutions and size of images, and the figures were changed.
In relation to the conclusion, in which we mentioned that our study could support animal tests and clinical trials, we really went far beyond what the work allows us to conclude. Based on this, we change the conclusion to: In summary, our results demonstrated for the first time the synergistic effect of OXY with chemotherapeutic agents in breast cancer cells, offering a new strategy for future animal studies.
Furthermore, in our laboratory, we are carrying out tests on animals using OXY and DOX, as well as other chemotherapy drugs and bioactive compounds related to different signaling pathways. The data is very promising, but will be included in another publication.
1- Figure 1 (A–C), it is impossible to read any numerical value. The figures are very small, the characters are microscopic, and the choice of gray scale is not very effective.
Author´s answer: The referee is correct and we apologize for that. The figures 1, 2 and 4 were changed. We believe that the figures are clear now.
2- Table 2, in my opinion, should be completely formatted
Author´s answer: The referee is correct and the suggestion was accepted. The table was formatted.
3- Figure 2 (C-D), it is almost impossible to appreciate the result
Author´s answer: The suggestion was accepted and figure 2 was changed. We believe that the figure is clear now.
4- Figure 4 (P and H), it is almost impossible to appreciate the result.
Author´s answer: The suggestion was accepted and figure 4 was changed. We believe that the figure is clear now.
