Bioactivity of Ionic Liquids Based on Valproate in SH-SY5Y Human Neuroblastoma Cell Line

Round 1

Reviewer 1 Report

Bioactivity of Ionic Liquids based on Valproate in SH-SY5Y 2 Human Neuroblastoma Cell Line
Manuscript ID: futurepharmacology- 1813519
Comments to the Authors

This paper investigates the potential of several ionic liquids (ILs) coupled with API Valproate as an antitumor agent. On evaluating the toxicity of the prepared ILs-API by MTT cell metabolic assay in human neuroblastoma SH-SY5Y and human primary Gingival Fibroblast (GF) cell lines, they showed inhibitory effects during the study period. The IL [C₂OHDMiM][VPA] demonstrated an outstanding antitumor activity against SH-SY5Y and lower activity against the non-neoplastic GF line, suggesting its potential as an antitumor agent.

Valproic acid is studied as a potential antitumor agent in neuroblastoma because of its ability to impair growth and induce differentiation in human neuroblastoma cells in harmless concentrations. A lot of current studies evaluate the physicochemical interactions of ionic liquids and valproate but there have been lesser reports on the bioactivity and toxicity of ILs-API. The significance of this paper is that the authors prepared several ILKS to be coupled with APL valproic acid and report their bioactivity through evaluation by MTT cell metabolic assay. They also evaluated the influence of the prepared compounds in different signaling pathways, which are important for the behavior of tumor cells.

The paper is technically sound and written in an easy-to-understand style. The authors provide a good background and introduction to the study. They have presented the technical terms and explained the experimental results in an easy-to-understand manner. No further experiments are needed, but I think this manuscript can potentially be improved through a few modifications as suggested below:

1.      The authors provide a very comprehensive introduction on why IL-API is being studied as an alternative cancer treatment. Can they comment more on how this cancer treatment alternative compares to currently existing cancer treatments?

2.      Currently reported data are for study period upto day 3. Is that the typical duration for studying cell toxicity? Can the authors comment on long term effects of ILs-API on human cells?

3.      It will be helpful if authors provide experimental details on how antitumor activity and cell viability are measured.

4.      In figure 3, it will be helpful if the authors describe the error bars in the data in the figure legend.

5.      It will be helpful if the authors can describe more what IC50 and LD50 mean.

Comments for author File: Comments.pdf

Author Response

Dear Editor and Referees,

Please find the following explanation, corrections and answers of your comments in this document.

In the manuscript figure 1 was removed as suggested redundant and other figures numerated accordingly. In addition, we have corrected some errors in the manuscript and SI (all additions are highlighted in the manuscript) and all punctual changes in SI too. In SI some spectra (1H and 13C NMR) were reprocessed in order to correct wrong assignment due mostly to erroneous calibration. In that case the whole figure name of the spectra is highlighted too as well as spectrum description.  

For more details please see joint pdf of corrected manuscript and SI in attachment.

Thank you for your collaboration.

Reviewer 1:

This paper investigates the potential of several ionic liquids (ILs) coupled with API Valproate as an antitumor agent. On evaluating the toxicity of the prepared ILs-API by MTT cell metabolic assay in human neuroblastoma SH-SY5Y and human primary Gingival Fibroblast (GF) cell lines, they showed inhibitory effects during the study period. The IL [C2OHDMiM][VPA] demonstrated an outstanding antitumor activity against SH-SY5Y and lower activity against the non-neoplastic GF line, suggesting its potential as an antitumor agent.

Valproic acid is studied as a potential antitumor agent in neuroblastoma because of its ability to impair growth and induce differentiation in human neuroblastoma cells in harmless concentrations. A lot of current studies evaluate the physicochemical interactions of ionic liquids and valproate but there have been lesser reports on the bioactivity and toxicity of ILs-API. The significance of this paper is that the authors prepared several ILKS to be coupled with APL valproic acid and report their bioactivity through evaluation by MTT cell metabolic assay. They also evaluated the influence of the prepared compounds in different signaling pathways, which are important for the behavior of tumor cells.

The paper is technically sound and written in an easy-to-understand style. The authors provide a good background and introduction to the study. They have presented the technical terms and explained the experimental results in an easy-to-understand manner. No further experiments are needed, but I think this manuscript can potentially be improved through a few modifications as suggested below:

1. The authors provide a very comprehensive introduction on why IL-API is being studied as an alternative cancer treatment. Can they comment more on how this cancer treatment alternative compares to currently existing cancer treatments?

Thank you for your comment and suggestion. Additional comment and explanation of contribution of ILs-API to currently existing challenges in cancer treatment was added, together with two more references (lines 86-92).

2. Currently reported data are for study period up to day 3. Is that the typical duration for studying cell toxicity? Can the authors comment on long term effects of ILs-API on human cells?

Ionic liquids are very diverse group of compounds with very tuneable physicochemical properties. The same apply to their toxicity. To the best of the authors knowledge and recent literature the toxic counterions, particularly cations are highly hydrophobic and very persistent to degradation. That is one the reason why we use a period up to day 3. The other reason we have assessed cell toxicity during 3 days because our goal was to evaluate possible acute effects of ILs. The cation used in this work, particularly [C2OHDMIM] are among the least toxic in earlier work.

3. It will be helpful if authors provide experimental details on how antitumor activity and cell viability are measured.

Cell viability was assessed by MTT assay, which is a method widely used to evaluate that parameter. It relies in the activity of mitochondrial enzymes, and only viable cells originate a positive result. The control used in the present work (gingival fibroblasts, non-tumour cell) allowed to understand if the obtained results could be tumour-specific, or if they were a consequence of a general cytotoxicity.

An explanation was introduced into manuscript (line 125-129):

4. In figure 3, it will be helpful if the authors describe the error bars in the data in the figure legend.

In Figure 2 (former figure 3) description of control and error bar was added (line 215-216).

5. It will be helpful if the authors can describe more what IC50 and LD50 mean.

The description was introduced in the manuscript (line 139-141).

IC50 indicates the drug concentration that is needed to decrease cellular metabolic activity by half.

LD50 indicates the drug concentration that is needed to decrease cell viability by half.

Author Response File: Author Response.pdf

Reviewer 2 Report

This work is aimed at obtaining (synthesis) of the ILs-API complex as potential therapeutic genes with antitumor properties based on valproate as an antitumor agent. The text of the MS is written qualitatively, clearly and will contain well-done illustrations of structure, yield and physical state of the synthesized ionic liquids based on VPA, general synthetic procedure of the ILs-API and cell viability in cultures.

 

The authors used classical methods of biotesting the complex on two cell cultures and showed low IC50 and LD50 values, inferring the potential of the prepared ILs-API as valid alternatives to cancer therapies.

 

In general, the authors presented a small work on synthesis of the ILs-API complex. The work is interesting, but I have some questions that I ask to answer

 

1) Why did the authors of the study choose SH-SY5Y and GF cell lines?

 

2) MTT test in my opinion is not very sensitive and has great limitations (conversion to formazan crystals depends on metabolic rate and number of mitochondria resulting in many known interferences). It would be great to show cell viability by ATP assay/ ATP assay is the fastest cell viability assay to use, the most sensitive, and is less prone to artifacts than other viability assays.

 

3) Why did the authors use only cultures and not model animals with genetically induced cancer? There are a lot of such models now. If this had been carried out, the conclusions would have sounded more reasonable.

Author Response

Dear Editor and Referees,

Please find the following explanation, corrections and answers of your comments in this document.

In the manuscript figure 1 was removed as suggested redundant and other figures numerated accordingly. In addition, we have corrected some errors in the manuscript and SI (all additions are highlighted in the manuscript) and all punctual changes in SI too. In SI some spectra (1H and 13C NMR) were reprocessed in order to correct wrong assignment due mostly to erroneous calibration. In that case the whole figure name of the spectra is highlighted too as well as spectrum description.  

For more details please see joint pdf of corrected manuscript and SI in attachment.

Thank you for your collaboration.

Reviewer 2:

This work is aimed at obtaining (synthesis) of the ILs-API complex as potential therapeutic genes with antitumor properties based on valproate as an antitumor agent. The text of the MS is written qualitatively, clearly and will contain well-done illustrations of structure, yield and physical state of the synthesized ionic liquids based on VPA, general synthetic procedure of the ILs-API and cell viability in cultures.

The authors used classical methods of biotesting the complex on two cell cultures and showed low IC50 and LD50 values, inferring the potential of the prepared ILs-API as valid alternatives to cancer therapies.

In general, the authors presented a small work on synthesis of the ILs-API complex. The work is interesting, but I have some questions that I ask to answer

1) Why did the authors of the study choose SH-SY5Y and GF cell lines?

SH-SY5Y cell line was chosen because it is a well-established neuroblastoma cell line. GF were chosen because we intended to have a primary non-tumor control, and also because considering an oral administration of a drug, gingival fibroblasts are cells that are potentially in contact with the active molecules.

2) MTT test in my opinion is not very sensitive and has great limitations (conversion to formazan crystals depends on metabolic rate and number of mitochondria resulting in many known interferences). It would be great to show cell viability by ATP assay/ ATP assay is the fastest cell viability assay to use, the most sensitive, and is less prone to artifacts than other viability assays.

Although MTT has some limitations, it is still widely used as a simple and effective method to assess cellular metabolic activity. Moreover, all cell cultures were kept in the same experimental conditions, and in all the experiments were included the corresponding negative and positive controls. Also, in the present work it was included a non-tumour control, in order to have a comparison point for the results obtained with tumour cells. Taken together, the experimental designed was developed in order to minimize possible interferences on MTT assay. Regarding ATP assay, although we recognize the value of that method, in the comparison between non-tumour and tumour cells may appear some significantly differences, since their ATP needs are quite different.

3) Why did the authors use only cultures and not model animals with genetically induced cancer? There are a lot of such models now. If this had been carried out, the conclusions would have sounded more reasonable.

We thank for the suggestion and agree that in vivo experiments would be of great interest. Nevertheless, the present work was directed to a more exploratory work, with the production and characterization of different ILs and the assessment of their in vitro biological activity. Also, and regarding bioethical issues and animal wellbeing animal models should only be used after all in vitro test are completed.

Author Response File: Author Response.pdf

Reviewer 3 Report

In spite of the significance of the topic, I see no description of the biological methods used in the study. How exactly the MTT assay was carried out? How the signaling pathways were assessed? Moreover, what is the difference between IC50 and LD50 provided in Table 2? Why there is no statistics, such as confidence intervals? The paper cannot be published without this information. There are also several additional drawbacks, which should be addressed more carefully.

1.       Figure 2 looks rather sloppy; I’d remove the pictures of the substances from it. Figure 1 looks redundant in comparison.

2.       Full names for the synthesized imidazolium API-ILs should be provided in the Introduction.

3.       I don’t understand the following sentence (p. 4): “The therapeutic dosage represents the concentration range of valproate in plasma, in which the concentration tested in both cell lines is within.” The concentrations provided in Table 1 are clearly beyond the therapeutic dosage.

4.       The significance of differences between the test substances and control for the fourth inhibitors should be indicated in Figure 3. The meaning of the error bars should be clarified.

Author Response

Dear Editor and Referees,

Please find the following explanation, corrections and answers of your comments in this document.

In the manuscript figure 1 was removed as suggested redundant and other figures numerated accordingly. In addition, we have corrected some errors in the manuscript and SI (all additions are highlighted in the manuscript) and all punctual changes in SI too. In SI some spectra (1H and 13C NMR) were reprocessed in order to correct wrong assignment due mostly to erroneous calibration. In that case the whole figure name of the spectra is highlighted too as well as spectrum description.  

For more details please see joint pdf of corrected manuscript and SI in attachment.

Thank you for your collaboration.

Reviewer 3:

In spite of the significance of the topic, I see no description of the biological methods used in the study. How exactly the MTT assay was carried out? How the signaling pathways were assessed? Moreover, what is the difference between IC50 and LD50 provided in Table 2? Why there is no statistics, such as confidence intervals? The paper cannot be published without this information. There are also several additional drawbacks, which should be addressed more carefully.

Regarding MTT assays we used the standard procedure. On the selected time points, cells were incubated MTT substrate for 3h. Then, cell layers were washed, formazan crystals were solubioized with DMSO and absorbance was quantified at 570nm. To evaluate the involvement of cell signalling pathways, cell cultures were mantained with the corresponding inhibitors, and cell response was evaluated at the defined time points. The differences between IC50 and LD50 are described below: IC50 indicates the drug concentration that is needed to decrease cellular metabolic activity by half. LD50 indicates the drug concentration that is needed to decrease cell viability by half. All the biological experiences were made with a minimum of 3 replicates and a maximum of 6 replicates. The work has statistics and there are confidence intervals in Figure 3 .

1. Figure 2 looks rather sloppy; I’d remove the pictures of the substances from it. Figure 1 looks redundant in comparison .

Thank you for your comment and suggestions. The figure 1 was removed as it really seems redundant. However, we decided to keep pictures and description of properties of prepared compounds due to easier visual distinction. In our long-term experience and work with OSIL-APIs although the physicochemical properties of the compounds (e.g., if they are ionic liquids (ILs), room temperature IL (RTILs) or organic salts (OSs)) do not directly correlate with the activity of the compounds they usually do so with the solubility and transportation due to effect of increase/decrease of total lipophilicity/hydrophilicity (counterion contribution). Those factors are then easier corelated in our mind to activity by retention of simple imagery. So, either they could be attributed just to change of physicochemical characteristics of API or, if not, some other more specific interaction can be easier spotted. In figure 2, in addition we detected and corrected a small structure error of [C2OHMIM] cation.

2. Full names for the synthesized imidazolium API-ILs should be provided in the Introduction.

The full IUPAC names of prepared compounds were introduced before more common names and abbreviations in the introduction part as well as in supporting info.

3. I don’t understand the following sentence (p. 4): “The therapeutic dosage represents the concentration range of valproate in plasma, in which the concentration tested in both cell lines is within.” The concentrations provided in Table 1 are clearly beyond the therapeutic dosage.

Thank you for your comment. The erroneous phrase was changed in manuscript (line 131-132).

4. The significance of differences between the test substances and control for the fourth inhibitors should be indicated in Figure 3. The meaning of the error bars should be clarified.

In Figure 2 (former figure 3) description of control and error bar was added (line 215-216).

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

no comments. Available for publication

Author Response

Dear Editor and Referees,

 

Please find in the attachment the pdf documents with respond and answers to your comments merged also with corrected manuscript and SI.

 

Thank you for your collaboration.

Yours sincerely,

Zeljko Petrovski

Author Response File: Author Response.pdf

Reviewer 3 Report

From the authors’ reply, it is still not clear to me how they determined both IC50 and LD50 by using only the MTT assay, which shows exactly the impact of a given substance on the activity of oxidoreductases in the cells being affected. Therefore, my question about the difference between IC50 and LD50 values remains unanswered. And the methodology should be described adequately in the paper, even if the procedure is standard (which, by the way, is not the case for ILs that often require additional controls in the MTT assay).

As for the statistics, in the revised manuscript there is no Figure 3, which, according to the authors, includes confidence intervals. If they mean Figure 2, the caption says the error bars correspond to standard deviations, not confidence intervals. In addition, the authors ignored the request to estimate the significance of differences between the experiments.

Thus, I again cannot suggest the acceptance of the paper in its current form.

Author Response

Dear Editor and Referees,

 

Please find in the attachment the pdf documents with respond and answers to your comments merged also with corrected manuscript and SI.

 

Thank you for your collaboration.

Yours sincerely,

Zeljko Petrovski

Author Response File: Author Response.pdf

Round 3

Reviewer 3 Report

I have to admit that I still have not understood how the authors managed to establish LD50 by using the MTT method. In this case, the absorbance reflects ONLY the activity of particular mitochondrial enzymes, but not the viability of the cells themselves. Other techniques should be used for distinguish between the cells, which are truly dead or alive. Therefore, I would ask the authors to provide more detailed description of the calculating procedures, together with references, in which the MTT assay is used for establishing both IC50 and LD50 values. This point is especially important due to the dramatic differences between some IC50 and LD50 provided in the manuscript. As for the paper by Sebaugh, there is no mentioning of LD50, only of relative and absolute IC50/EC50 values.

Author Response

Dear referee,

 

Please see document in annex.

Thank you for your collaboration.

 

Yours sincerely,

Zeljko Petrovski

 

Author Response File: Author Response.pdf