Establishment of Acquired Cisplatin Resistance in Ovarian Cancer Cell Lines Characterized by Enriched Metastatic Properties with Increased Twist Expression

Round 1

Reviewer 1 Report

Upon reading the manuscript Bahar et al. as well as reviewers and authors comments (revision round one) my opinion is that the authors improved the quality of the first manuscript version.

Unfortunately, my honest opinion is that the manuscript is not presenting any novel data. The authors claim that underlying mechanisms upon Twist knockdown were investigated. Actually that is not true since the experiments performed and data obtained are already well-known effects (drug induced EMT, Twist regulated EMT, PARP regulated EMT, Twist correlation with expression of DNA repair proteins) and pretty much investigated superficially (Twist knockdown regulation of constitutive expression of some cell death, ER stress or DNA repair proteins). The manuscript would be novel if authors investigated in more detail Twist regulated either ER stress or DNA repair mechanism in correlation with cells response to cDDP. These regulatory mechanisms are not known in the literature. Moreover, nowadays it is not enough only to check the expression of several proteins at constitutive level in other to assume that they are involved in cell death, ER stress, DNA repair and cDDP resistance since it is known that functional assay can result with opposite findings especially in drug resistant cells.

With other words, I do not think that observed effects on expression of Bcl-2, BAX (the Figure 8B and D are not convincing without densitometry and SD values for ER stress proteins) upon Twist knockdown are novelties for the field. Moreover, why parental cell protein extracts are avoided in Figures 6-8? It will be interesting to see how Twist knockdown really changed the protein expression of explored proteins.

I am encouraging authors to investigate in more details one of the signalling pathway I mentioned above and publish more novel data.

Author Response

We would like to express our appreciation for the reviewer’s valuable comment to improve manuscript quality. We have investigated ER stress signaling pathway with more data. Please see Figure 9 and Figure 10. In addition, according reviewer suggestion we have included parental cells in experiment for Figure 6 to 10.

Reviewer 2 Report

The research is well conducted in the intent, demonstration of the results and discussion. The mechanism of chemoresistance represents one of the most important factors for the survival of cancer patients suffering from epithelial carcinoma of the ovary. Understanding the various steps of this mechanism represents an important value. The new method proposed "Twist" seems to consolidate the previous analyzes with a sort of confirmation of what it is thought about the mechanism of chemoresistance.

Some general recomandation:

• I ask the authors if they were able to perform the same experiments with cells from ovarian cancer patients. cell lines have their limits in translation into clinical practice. If they have any experience with patient cell cultures I would ask to discuss it, otherwise point it out as one of the limitations of the study in discussion.
• it is recommended to add mean and SD in the text and figures

and

Line 145-148: Comments are generally avoided in the results. If not strictly necessary, as it seems to me, I would take them off or put them in discussion.

Line 206-209: the same comment of the line 145.

Author Response

• I ask the authors if they were able to perform the same experiments with cells from ovarian cancer patients. cell lines have their limits in translation into clinical practice. If they have any experience with patient cell cultures I would ask to discuss it, otherwise point it out as one of the limitations of the study in discussion.

Reply: We have point out this limitation in discussion section. Lines 397 to 399

• it is recommended to add mean and SD in the text and figures

Reply: Added mean and SD in the text and figures. (e.g line 110 to 129, Figure 2)

and

Line 145-148: Comments are generally avoided in the results. If not strictly necessary, as it seems to me, I would take them off or put them in discussion.

Reply: Shifted that part to discussion section. Line 327 to 330

Line 206-209: the same comment of the line 145.

Reply: Moved that part to discussion section. Line 336 to 339

Reviewer 3 Report

Dear authors

the paper describes two novel cell lines resistant to cisplatin by using pulse dosing and stepwise dose escalation methods for a duration of eight months to mimic what happen in clinic. The resistant clones showed many characteristics of metastatic potential that in part could be ascribed to TWIST expression.

This is a resubmission in which the authors add more experiments to reply to referees. I believe that the paper improved a lot. 

Regarding TWIST, there are other articles that demonstrated the role of this protein in drug resistant in ovarian cancer. I suggest to the author to discuss the following papers:

Author Response

 We have discussed the Twist and drug resistance in view of the mentioned articles. Lines 383 to 388.

Round 2

Reviewer 1 Report

Upon revision the manuscript is improved. There are several things which need to be improved before final acceptance of the manuscript.

1) Are parental cells in Figure 6-10 transfected with negative control (siNC) or non-transfected cells? The main reason I am asking they question is that transfection by itself can change transcription of many genes having an effect on downstream events. If the cells are not transfected then the authors need to point the readers why not. 

2) I am not sure that I understand this statement Line 52...The process of acquiring cisplatin resistance is not fully understood, but is believed to develop through cross-resistance to a wide range of chemotherapeutic drugs with different mechanisms of action...

The point is that acquired resistance can develop upon treatment with only one drug but the events upon can influence cell to become cross-resistant to several different type of drugs...please write this sentence more clearly and correct.

3) Line 59 upregulation of multidrug resistance (MDR) protein...is this really correct? MDR protein is another name for P-gp? Or do you mean proteins from MDR family?

4) Line 380..change ovarinn in ovarian

5) Fig 2 description...please add one sentence what is marked with circular markers on photos (it will be easier for a reader to understand what authors wanted to point out without searching in the text)

Author Response

Upon revision the manuscript is improved. There are several things which need to be improved before final acceptance of the manuscript.

• Are parental cells in Figure 6-10 transfected with negative control (siNC) or non-transfected cells? The main reason I am asking they question is that transfection by itself can change transcription of many genes having an effect on downstream events. If the cells are not transfected then the authors need to point the readers why not. 

Reply: Thank you very much for your valuable comments and suggestion that help us to improve manuscript quality.

We have used non-transfected parental cell in our experiment, in order to compare re-sanitization capacity of CisR cells by Twist knockdown (Elodie E. Noel et al Am J Pathol. 2010 Jun; 176(6): 2607–2615). Line 244 to 245. In general, negative control exhibits alien sequence and non-lethal properties as to not affect mRNA and protein expression levels due to non-target design.

2) I am not sure that I understand this statement Line 52...The process of acquiring cisplatin resistance is not fully understood, but is believed to develop through cross-resistance to a wide range of chemotherapeutic drugs with different mechanisms of action...

The point is that acquired resistance can develop upon treatment with only one drug but the events upon can influence cell to become cross-resistant to several different type of drugs...please write this sentence more clearly and correct.

Reply: We have revised the sentence with correct meaning as “but it is believed that tumors resistances to cisplatin are also resistance to the other platinum drugs”. Line 54 to 55.

3) Line 59 upregulation of multidrug resistance (MDR) protein...is this really correct? MDR protein is another name for P-gp? Or do you mean proteins from MDR family?

Reply: We have revised the sentence with correct meaning as “cisplatin-resistant oral squamous cell carcinoma cells upregulate the activity of multidrug resistance protein 1 (MDR1) or breast cancer resistance protein (BCRP) associated with EMT”. Line 60 to 62.

4) Line 380..change ovarinn in ovarian

Reply: We have revised.

5) Fig 2 description...please add one sentence what is marked with circular markers on photos (it will be easier for a reader to understand what authors wanted to point out without searching in the text)

Reply: We have point out the circle on images as “macronucleoli” in figure 2.

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.

Round 1

Reviewer 1 Report

General Concerns

Improving current models for the analysis of chemoresistance will always be welcome by the research community. Regardless of the method of delivery used herein, for the most part, the results support previous findings made by others. The authors fail to demonstrate what is so markedly different/novel about their results that move the field significantly forward.  The endpoints measured are straight forward. The interpretation of the data is relatively in line with the outcome, albeit the terminology used implies more than what is being observed (ex. wound healing). Nevertheless, the authors focused on superficial endpoints that hint to changes in multiple processes associated with platinum-based resistance but there were no dramatic differences between the two systems. This lack of marked difference reduces the overall enthusiasm.

In general, there are numerous grammatical errors that force the reader to assume intent. It would be helpful to have someone proofread the manuscript to eliminate these issues, so the reader can focus on the scientific content and not guessing at what was intended.

There seems to be an inordinate amount of discussion in the results section which should be reserved for the discussion section. An example of this is lines 254-263.

The advantage of the ANN model system is not clearly shown. The relevance of the neurons in the hidden layer needs to be better explained and justified why it is superior to what has been shown historically. Secondly, if it requires such a sensitive analysis to detect differences, it brings into question whether they are physiologically relevant.

Given the authors are talking about cells changing in size the authors should refer to cell proliferation as opposed to cell growth when measuring cell number.

Based on the adhesion assay the authors are implying that the resistant cells are more adherent yet at the same time they are arguing that they are more metastatic. In the case of ovarian cancer, the metastatic lesions are distal to what the perceived sites of origin – if the cells are so adherent how are they migrating to distant areas?

There is no justification for the specific cell lines chosen. This is especially important that SKOV3 and cells were shown by Domcke et al (2013) to be least like high-grade ovarian cancer. OV90 cells were not shown to be much better as a model system.

Specific issues

The grammatical errors are too numerous to list but examples are provided below.

Lines 144, 145, 152, 155 and more – Based on the method of analysis used it does not seem that speed is the appropriate word for the outcome. It is assumed the authors were trying to demonstrate that there was an increase in the number of cell migrating.

Line 150 – In invasion assay, CisR cells showed higher invasion….. – should read The invasion assay showed a greater level of ….or increased levels of…

Lines 262-263 – An awkward sentence and needs to be re-written.

Line 290 - Two different size fonts.

Line 292 – Please define strength of drug resistance.

Reviewer 2 Report

Well designed and written study. Could be accepted for publication.

Reviewer 3 Report

In the present manuscript entitle: „Establishment of Acquired Cisplatin Resistance in Ovarian Cancer Cell Lines Characterized by Enriched Metastatic Properties Associated with Epithelial-Mesenchymal Transition Signature, DNA Repair Alteration and Repression of Endoplasmic Reticulum Stress-mediated Cell Death” Bahar and colleagues generate cisplatin-resistant ovarian cancer cell lines by pulse dosing and stepwise cisplatin dose escalation methods for a period of eight months. They propose to elucidate the mechanism which is responsible for the acquired cisplatin resistance and perform plenty of different experiments for instance measurements of DNA repair proteins, EMT markers, and determination of ER stress-mediated cell death. In general, the manuscript is well prepared and contains a lot of experimental data but it does not fulfill the proposed idea of identification of the mechanism of cisplatin resistance. In fact, the authors finally conclude that cisplatin induces an EMT program in the cells associated with an increased cellular migration, reduced apoptosis, accelerated DNA repair and finally resistance towards cisplatin. These findings are not new and have been published for other cancer entities as well as for ovarian tumor cells elsewhere: “J Cell Biochem. 2011 Oct;112(10):2850-64. doi: 10.1002/jcb.23199” or “Mol Cancer. 2013 Mar 27;12:24. doi: 10.1186/1476-4598-12-24.” Even reviews dealing with the association of EMT with resistance are available :” Hum Pathol. 2013 Nov;44(11):2373-84. doi: 10.1016/j.humpath.2013.05.001. Epub 2013 Jul 11.” Thus, the manuscript does not add novel aspects to chemoresistance towards cisplatin and it is questionable if the degree of novelty of the manuscript is sufficient to be published in Cancers with an impact of 6.1? Maybe the manuscript can be shifted to another MDPI journal with a lower impact factor after thorough modifications! In the present form, it should be rejected!    

Major concerns:

• Are there morphological differences detectable between SKOV-3/CisR1 and SKOV-3/CisR2 cells?
• Are the cells in figure 2A are treated with a specific dye or why do they appear in blue color?
• Line 111: “to inhibit 50% of cells” => proliferation or death?
• Line 112: how many repetitions did you do of each experiment? Is it n=1? What is the SD?
• Lines 112 to 127: you don´t have to mention each concentration required to obtain a 50% growth inhibition. It is sufficient to indicate the fold changes as you did in lines 125 and 126. Otherwise, it is difficult to read and follow.
• Please indicate n in the legend to figure.
• What do the microscopic pictures in figure 3 add? They are too small to recognize something. Additionally, scale bars are missing.  
• Figure 3: Are SD or SE indicated in the figure? Please provide some information. Please indicate n in the legend to figure 3.
• The differences between your migration and invasion assay is not clear. In the “Materials and Methods” section for both assays, you write: ”cells attached to the bottom side of the membrane were fixed by methanol, stained with 0.1% crystal violet, dried, and photographed under a light microscope”. (lines 425, 426 and 446, 447). How did you differentiate between invasion and migration?
• Conclusion: “this study’s findings could allow us to understand the molecular mechanism of complex acquired cisplatin resistance, and develop a new strategy to overcome cisplatin-resistant ovarian cancer.” => this is a complete over-interpretation of your results since you offer no new targets in the context of resistance formation or any helpful information for a better understanding.
• In wound healing assay: in some cases you indicate % of wound closure and then area in μm²/h? Why?
• Line 210: you refer to figure 5?
• Lines 203 and 204: what are the exact functions of PARP1 and XRCC1 in DNA repair? Please explain in one sentence precisely and don´t say “they play an important role”.
• Line 212: Do you refer to figure 5?
• Figure 5D: The standard deviations for protein expression relative to beta-actin are very small. What is the number of repetitions of experiments and how much data were included in your calculations? Please indicate.
• Figure 5E: It is evident that in resistant cell lines DNA repair is enhanced whereas apoptosis-related proteins are reduced. What kind of new information does figure 5E add? Delete it!
• Figure 6 could appear in a review but is not appropriate for a research article: Please delete it!
• Can you revert a cisplatin resistance in both cell entities by inhibition of TF twist either by knockdown with siRNA or a small molecule inhibitor? This would be an interesting approach that would enhance the quality of the manuscript.
• Why did you choose fibronectin for your adhesion assays and not any other substrate of the extracellular matrix? Which tumor cell-expressed adhesion receptors are responsible for binding to fibronectin?
• There is no information concerning statistics… why?
• In the discussion section, you are writing: “The results of our study are also consistent with previous studies because the significantly increased level of XRCC1 and PARP1 correlated with cisplatin resistance in both SKOV-3 and OV-90.” So what is the news your present study provides compared to older results?
• The discussion section is more an explanation and repetition of the obtained results than a critical debate in the context of the current literature.
• The last sentence in the discussion says it all: “we speculate that EMT contributes to the cisplatin resistance mechanism of OC cells” => this has been shown by numerous other groups.

Minor concerns:  

• Line 91: please provide the units for cisplatin concentrations applied
• Line 419: 8.0 μm polycarbonate => pore is missing
• Lines 529, 530,532,533,534 and 536: you do not have to indicate Dallas, TX, USA every time for Santa Cruz. One time is enough.
• Line 290: “within the body”: the font is different
• Line 279: what is N/R-cadherin?
• Line 231: 50 µM and not µm.
• Line 326: have been and not “has been”.
• Line 336: full stop is missing.