Indole-3-Carbinol, a Phytochemical Aryl Hydrocarbon Receptor-Ligand, Induces the mRNA Overexpression of UBE2L3 and Cell Proliferation Arrest

Round 1

Reviewer 1 Report

The authors herein report the effects of I3C on held cell line and on real life sample.

The experimental workflow is too simple. Analyses of two genes is too reductive. Moreover I do not understand the use of endpoint PCR rather than qPCR. This issue need to be assessed.

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Reviewer 2 Report

Overall, the manuscript is interesting and offers enough information to be publised in the jorunal. Figure 6 is really illustrative and demosntrate the hypothetical model for the I3C mechanism on Hela cells. However, there are few comments that need to be addressed before publication.

• Statistical analysis is missing from some graphs. Please, include which type of test is performed along with p-values.
• Please discuss the impact of I3C on other type of cells. 
• How can you deliver it to the cell to achieve a significant effect? Is literature available regardign this point?
• Teh addition of complementary data in this manuscript to prove your hypothesis is encouraging.

Author Response

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Reviewer 3 Report

          The article has an interesting topic, but must be improved.

1. in the abstract section, authors write: Cell culture and patients’ samples were used to determine the effect of indole-3-carbinol (I3C) effect over cells viability, apoptosis, cell proliferation and mRNA levels of 45
   UBE2L3 and CYP1A1" how this observation was made  in patients' sample, the effects of I3C regarding viability, apoptosis, cell proliferation? The authors did not incubate cells from patients with I3C.
2. why it was used BNF, a  anticancer agent  mammary carcinoma cells, one for cervical cancer do not exist?
3. a CC-Arh exist?
4. a few samples  from patients are irrelevant for solid conclusions
5. cells isolated from patients with CC, CIN and healthy volunteers should be incubated with I3C
6. why Hella cells have been used?

Author Response

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Reviewer 4 Report

Arellano-Gutiérrez et al. report on the effect of I3C, an AhR agonist precursor, on HeLa cells proliferation and apoptosis as well as AhR target genes CYP1A1 and UBE2L3 expression levels. This informs on the mechanism by which I3C may affect HPV-induced cervical cancer through the biological activity of viral oncoproteins E6 and E7.

The manuscript is interesting but has a few severe shortcomings. The apoptosis assay is missing proper controls to draw any conclusions from it. Same goes for the 5% increase in mRNA levels between I3C and BNF which are not statistically significant and cannot be the basis for any conclusions. Therefore, a lot of the authors conclusions seem largely overinterpreted. I advise for major revisions before publication.

 

Major

 

L149 – the concentrations of I3C, BNF or CPT are not the same in different assays. Is there a specific reason for this? Are the results comparable?

L212 – I think the authors mean EC50 instead of IC50. Furthermore, I fail to get how the authors calculated a value of 0.1 uM from their results. An EC50 value should also be accompanied with an error, which should be sizeable given the data.

L261 – were the histograms somehow normalized to untreated cells? I fail to understand the point of the assay without an untreated negative control. Furthermore, there are clear visual differences between drugs in terms of apoptotic/non-apoptotic cell ratios and late/early apoptotic cell ratios, the authors should elaborate on this.

L343 – The 5% increase in UBE2L3 mRNA level by I3C compared to BNF is not statistically relevant by any means. No conclusions can be drawn from these results.

L363 – I am not sure how the conclusion of no change in cells apoptosis is drawn. Comparison of different agonists at different concentrations without negative controls and normalization is not ground for that conclusion.

 

Minor

 

L63 – LCR stands for locus control region

L73 – a piece of mechanistic information about E6 is missing. E6/E6AP binds UBE3A E2 ubiquitin-conjugating enzyme thereby leading to ubiquitination of p53 (and other substrates) which is targeted to the proteasome.

L192 – what kind of repeats were used? Technical or biological?

L196 – the chi2 tests results are not presented anywhere in the manuscript

L209 – were p-values calculated between 0 and >250 uM I3C levels?

L226 – which mRNA panel B quantifies is not indicated

L234 – Surprisingly, there is less target mRNA increase at 200 uM I3C than at 100 uM. This trend is interesting and should be discussed further.

L299 – technically there is a decrease of 1.5-fold, or approx. 33% decrease.

L319 – do the authors think that I3C condensates into AhR agonists or that I3C binds AhR? This is not clear and should be explained in the introduction as well. If I3C is a precursor, agonists of AhR should be identified in the culture medium by chemical means or mass spectrometry.

Author Response

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Round 2

Reviewer 1 Report

Dear authors,

In my first round of revision, I strongly suggested you increase the number of genes analyses and, in particular,  analyze real-life samples via qRT-PCR.

An endpoint PCR followed by densitometric analyses as you describe do not represent a measurement of expression level. I regret having to report such as issue which is mandatorily dealing with gene expression. Or you have to remove this part from the manuscript (and obviously you have to improve with other data) or you have to perform a  semiquantitative PCRfollowed by agarose gel. 

Without this, these measurements are not valid.

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Reviewer 3 Report

The authors have improved the article

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Reviewer 4 Report

Thank you for carefully taking into account all my comments and adjusting the scope of your manuscript accordingly. Please find below a few remaining questions that you should again take into account before publication.

• L149 – the concentrations of I3C, BNF or CPT are not the same in different assays. Is there a specific reason for this? Are the results comparable? - What I meant by this was rather the comparison between compounds than the comparison between different I3C concentrations. Are EC50s or Kds known for I3C, BNF and CPT? How was concentration of each compound chosen and are they comparable?

"Moreover, we calculated a half-maximal effective concentration (EC50) for I3C of about 500 µM in HeLa cells (R2 = 0.9342.). However, all experiments were performed with lower concentrations of I3C than those that activate AhR."

• This is slightly confusing, if all experiments are done much below I3C's EC50, where do the observed effects come from? Are we looking at off-AhR effects?

Apoptosis assays - I still think that there are some interesting features in the distributions that should be explained. Please elaborate more on this.

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Round 3

Reviewer 1 Report

Dear authors, once again I need to discuss the endpoint PCR you presented. In such type of analysis you cannot present a gel showing the result of the reaction at 40 cycles of PCR. You have to present a gel showing multiple loading of samples taken at different cycles(eg. 5-10-15-20...40) so as to appreciate in the dynamic range of amplification  the differences during exponential phase.

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