In Vitro Anti-Breast Cancer Effects of Tamarix aphylla-Derived Quercetin and In Silico Insights into Its Targeting of PIP4K2A

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is an interesting, multidisciplinary approach to the evaluation of the anti-cancer activity of a natural product. While the methodology appears sound, the presentation suffers from a lack of attention to detail.

This study presents results for Quercetin, as an anti-cancer agent. The compound was extracted from Tamarix aphylla,but little detail is given as to the purification method – I assume it was preparative HPLC. Since the crude extract contains multiple components, more detailed analysis is needed to confirm the purity and identity of the Quercertin.  At least mass spec, NMR or elemental analysis is needed. FTIR and UV analysis can only at best identify the compound class.

The crystal structure of PIP4K2A had a co-crystallised native ligand but no details are given as to what this ligand is and what its function is. A comparison of the original native ligand and the re-docked ligand would have been useful.

 

Fig 2. Legend is wrong. B and C are transposed. The ligand is green sticks not cyan. The ligand in A is not the same as the ligand in B although they are both meant to be the native ligand! Table 2 says there are no H-bonds to the native ligand yet they are clearly shown in Fig2C.

 

Fig3. Again, legend to wrong. Quercetin is green not pink in B not C. Figures A and C should be combined.

Fig 4. RMSD plot shows quercetin undergoes movement in the binding pocket. Does it also change conformation? An overlay of the structure of quercetin before and after dynamics would show this. What about the RMSD of the protein? Both ligands induce the same protein RMSD. What is the significance of this? What does the RMSF plot tell us? The protein has different RMSF profile for the two ligands and the interacting residues are different. The significance of the pink and blue regions is not explained. The bottom graphs serve no purpose.

Fig5 and 6. There is no indication as to which ligand each figure refers to. Initially I thought Fig5 was the native ligand but it has strong H-bonds with ASN-198 and Val-199. In the text and in Fig 4 we are told the native ligand has no H-bonds!

Fig7 Suddenly we are introduced to 8C8C. I see very little difference between the two ligands.

Fig 8 needs to be explained. The free energy scale for both are the same. The native ligand seems to have two, well defined minima, while quercetin and one broad energy valley.

 

Minor comments

L121 What are non-catalytic water molecules.

L495 plunge to an astonishing 0.025-fold compared to controls ---- 0.025-fold what?

L502 The conclusion that quercetin locks the ATP pocket is not supported by the MD results. The RMSD and RMSF of the protein with  both ligands is very similar.

A space should be left between a number and the units.

Since the free energy was calculated over a range of times the error should be given.  

I am unfamiliar with the units mum/h and  200 \ \muL and what the two \ mean

L193 The total RNA was isolated from what?

 

 

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

It integrates computational simulation and in vitro biological verification to systematically demonstrate the dual anti-breast cancer metastatic mechanism of quercetin. The results are solid, innovative, and provide a promising therapeutic candidate for targeted breast cancer treatment. Here are my opinions:

 

Four major flavonoids were identified by matching chromatographic peaks with commercial reference standards, including one unknown flavonoid at 3.94 min, rutin, quercetin and kaempferol. Please elaborate on the rationale for selecting quercetin rather than the other three compounds for subsequent functional and mechanistic investigations.

 

Quercetin forms stable canonical hydrogen bonds with backbone atoms of two critical hinge residues VAL199 and ASN198, with high occupancy values of 91% and 87%, respectively. It is worth questioning whether mutagenesis of these two amino acids would alter the docking binding threshold and binding affinity of quercetin. Supplementary docking simulations using mutant protein models are suggested to validate their indispensable roles in ligand binding.

 

Cytotoxicity assays were only performed on MDA-MB-231 and MCF7 breast cancer cell lines. It is unclear whether the authors have tested the cytotoxic effect of quercetin on normal mammary epithelial cell lines such as MCF-10A.

 

Scale bar information for panel A in Figure 10 should be added to the figure legend.

 

According to the scratch images in Figure 10A, the group treated with higher concentrations of quercetin appears to exhibit faster cell migration, which is inconsistent with the quantitative statistical results presented in Figure 10B. The authors are requested to provide a reasonable explanation for this discrepancy.

 

Panel A of Figure 11 only contains horizontal axis labels without vertical axis titles; corresponding vertical axis labels need to be supplemented.

 

It is unclear how many individual cells were included for statistical analysis in Figure 11B. If only a small number of cells were quantified, the reliability and persuasiveness of these statistical results would be insufficient. The exact cell count for each group should be provided in the figure legend or text.

 

It is not specified which cell line was used to obtain the qPCR data in Figure 12. The authors are suggested to supplement the corresponding qPCR results of both MDA-MB-231 and MCF7 cell lines for comprehensive comparison.

 

The manuscript lacks definitive genetic proof that Tamarix aphylla-derived quercetin exerts anti-breast cancer effects via targeting PIP4K2A. To validate this core claim in the title, the authors are required to perform PIP4K2A knockout and overexpression rescue experiments, followed by proliferation and migration functional detection with quercetin administration. Current data cannot support the proposed PIP4K2A-dependent mechanism.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed all my concerns. The manuscript, is I think now much stronger.

i do note an error on line 283 where docking score is reported as 10.0 but everywhere else is given as 10.77

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have greatly improved the quality of the manuscript. I recommend acceptance in its present form.