Phenanthroindolizidine Alkaloids Isolated from Tylophora ovata as Potent Inhibitors of Inflammation, Spheroid Growth, and Invasion of Triple-Negative Breast Cancer

Round 1

Reviewer 1 Report

This manuscript first explored structure-activity relationships between phenanthroindolizidine alkaloids (PAs)-derived compounds and their functions, e.g., NFκB inhibition and tumor cell cytotoxicity. Next, the authors evaluated the consequence and cause of NFκB inhibition by compound 1s. They found that compound 1s was capable of inhibiting HIF activity and stabilizing inhibitor of NFκB (IκBα) under both normoxia and hypoxia conditions. They further established the TNBC cell line MDA-MB-231 3D spheroid for the inhibitory effects of compound 1(1s) on tumor cell invasive activities. The anti-cancer  (decreased cell viability) effect of compound 1s was due to cell cycle arrest at the G2/M phase. They thought that PA-derived compounds (1/1s) might serve as anti-cancer agents with multiple target sites to combat inflammatory and hypoxia-driven cancers with a different mode of action than paclitaxel. Overall, the findings are interesting with some concerns as follows:

1. There were 4 repetitions of Table 1. Three of them should be removed.

2. Lots of expressions were in Germany throughout the entire manuscript. These should be thoroughly converted into English for consistency and easy understanding.

3. What was the logical reason to chemically synthesize the 1s compound? Isn't it exactly the same structure as compound 1? If they might be different (due to impurity?), why was compound 1 used only for results in Fig. 1-5, but only compound 1s was used in Fig. 6-10? While compounds 1 and 1s worked in Fig. 1-5, it doesn't mean that they would work in Fig. 6-10.

4. I don't see how the cytotoxicity of PAs was done in Materials and Methods where I could only find 2D or 3D viability assays, which only tell you the levels of viable cells but not those of cytotoxicity. A reduced number of viable cells might not necessarily be due to increased cell death. It was likely to be an inhibition of cell growth.

5. How was the quantification in Fig. 5a calculated? Was it based on the IF staining of the spheroids in Fig. 5b-4d? Please clearly describe that.

6. The inhibition of HIF activity by compound 1s was not necessarily due to NFκB or stabilization of TNFα-suppressed IκBα in Fig. 7 and 8 unless rescue assays were performed in which the compound 1s-inhibited HIF activities could be restored upon NFκB constitutive activation/overexpression or IκBα silencing.

7. I don't see dose-dependent reversions in Fig. 8a-8d.

8. Although blockade efficacy of nuclear factor NFκB and cytotoxicity by compounds 1(1s) were not totally at the same level, could inhibition of NFκB is, at least partially if not all, involved in the pathway leading to the compound-induced cytotoxicity, cell cycle arrest, or even spheroid invasion inhibition?

Author Response

The authors would like to thank the reviewers for the valuable feedback.

Please see the attachment for a detailed point-by-point response.

Author Response File: Author Response.pdf

Reviewer 2 Report

The English language in the overall manuscript is not very clear and a bit confusing. Therefore, I would recommend the authors to re-write the manuscript specially the procedures/results section to make it more understandable and readable. Also, the sentence construction is not appropriate. So, I would like to review this manuscript again after proper editing.

This work looks promising and the manuscript may be accepted for publication, if the authors properly address all the concerns raised.  

Some key points to be mentioned are as follows:

1. H2O should H₂O and so on…

2. Re-write Line no 911

3. I would recommend authors to perform Angiogenesis assay.

4. In Fig 2  (error bars are missing)

5. Line 312 typo error.

6. On page 5 of 43, both tables appears to be similar. Please check?

Table 1 has been repeated 4 times in the article. Why? Also, why variable MDA-MB231 (superscripts 1, 2, 3) were chosen for different time points? Where is the superscript 5 shown as per caption?

7. What does scale bar represent in Fig 6?

8. Fig 10 (error bars are missing).

9. What does this sentence means - Fehler! Verweisquelle konnte nicht gefunden warden?

10. Authors should show the graphs corresponding to the cytotoxicity of 1, 2, 3, 4, 5, 6 and Paclitaxel.

11. I would recommend authors to include clonogenic assay and scratch assay.  

12. Increase the resolution of figures.

13. Show the in-vitro stability profile of 1/1(s) at different pH and temperatures. 

Author Response

The authors would like to thank for the valuable suggestions. Most parts of the material section have been re-written.

Attached you will a find a detailed point-by-point response.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

1. The explanation for why they used a chemically synthesized compound in comparison should be put at the beginning of the result section for a better understanding.

2. If the authors only did viability assays (cell confluency) without really performing a cytotoxicity assay (e.g., PI/annexin V flow cytometry or TUNEL assay), they should not put "cytotoxicity of PAs" because it would confuse readers. They should just describe it as the viability of PAs unless further perform a cytotoxicity assay. Observing increased DNA fragmentation or the PA effect on the K562 cell line can not justify the cytotoxicity as the authors interpreted.

3. The authors' response to my comment #6 should be added to the discussion for Fig. 7 and 8.

4. What I meant in my previous comment #7 was that I could not see "dose-dependent" reversions in Fig. 8a-8d as the authors described in the title of Fig. 8, which defeats the specificity of PA effects on IκBα stabilization. How would the authors address this issue?

5. The authors' response to my comment #8 should also be integrated and added to the discussion for Fig. 7 and 8.

Author Response

Please, see the attachment.

Author Response File: Author Response.docx

Round 3

Reviewer 1 Report

I only have one concern left after the authors properly addressed my comments. Although I can see a dose-dependent manner in the representative western image (Fig. 8d), the statistical analysis in Fig. 8c showed a significant difference only in the treatment with a 20 nM compound. I still couldn’t see “dose-dependent" reversions in Fig. 8c among treatments with different concentrations of compound 1s. Since the results do not support the conclusions made by the authors, perhaps the authors could just show the results of 20 nM treatment for both hypoxic and normoxic conditions and delete the “dose-dependent” for the title of Fig. 8 and the heading of subsection 2.7.

Author Response

The authors would like to thank for the valuable suggestion.

We have adjusted figure 8 accordingly focussing now exclusively on a compound concentration of 20 nM and corrected the title and parts of the content of chapter 2.7  as highlighted in green.

Round 4

Reviewer 1 Report

I fave no further questions.

Author Response

The authors would like to thank the reviewer for the helpful suggestions.