Fucoxanthinol Promotes Apoptosis in MCF-7 and MDA-MB-231 Cells by Attenuating Laminins–Integrins Axis

Round 1

Reviewer 1 Report

Reviewer’s comments:

In the current article entitled “Fucoxanthinol Promotes Apoptosis in MCF-7 and MDA-MB-2 231 Cells by Attenuating Core Signaling Pathways” authors have tried to find the mechanism of FxOH mediated cell death in MCF-7 and MDA-MB-231 cell lines through transcriptomic profiling. The English language is fine, just need little improvement, some places sentences are not written properly. The manuscript is written properly however I have following comments:

The role of FxOH has been already checked and reported in MCF-7 and MD-MB-231 cell lines, what is the main purpose of the study? Is it just to find out the pathway/s involved in FxOH mediated cytotoxic effect?

Line 49: Do the authors mean “BC is the leading cause of cancer death in women”?

Lines 79-81: Please read the lines and write in proper meaningful way.

Section 2.2, 2.3, 2.4, 2.5, 2.7 and 2.8: Please mention the exact cell numbers used in each assay, not just the number of cells/ml. Current representation is misleading since the cell numbers change depending on change in the media volume OR provide additional information about the volume of media used for each plate type.

Lines 245-263: Are those observations recorded on Day 2 for both cell type? Please clarify.

Figure 2: Please provide scale bars for Figure 2B.

Section 3.5: Why authors checked the expression levels of many proteins here when those were not commonly regulated upon FxOH treatment between both the cell lines? Please provide the explanation.

Line 376-379: Sentence is not fully completed, still pending.

Section 3.6: Before stating integrin-β1 or integrin-β4 KD studies, please mention why authors picked them from the proteomic analysis, what is the significance of integrins in the context of cancer progression.

Figure 7: Western Blots are not clear; many proteins are not even visible on the blots. Please provide higher exposure for all those proteins which include- Fibronectin, Integrin-β6, Cyclin B1, CCR1, CCR4, CCR7, PRLR, pAKT(Ser⁴⁷³) and both NF-kB blots.

Provide enough β-actin, GAPDH or any loading controls for respective WB panels. Presenting just one loading control for so many blots is not acceptable.

Line 477: Spelling mistake in “induction”. Please correct.

General Comments: Any cytotoxic or cell-static drug will alter multiple pathways which are essentially related to cell cycle, replication, mTOR, MAPK, TGF-β in some context of cancer biology. At the end, authors need to conclude and highlight the precise mechanism (specific pathway/s by which FxOH works). I guess, authors primarily found laminins-integrins axis, if that is the case, please highlight precisely and accordingly the title of the paper may need to be modified.

Author Response

Reviewer’s comments:

In the current article entitled “Fucoxanthinol Promotes Apoptosis in MCF-7 and MDA-MB-2 231 Cells by Attenuating Core Signaling Pathways” authors have tried to find the mechanism of FxOH mediated cell death in MCF-7 and MDA-MB-231 cell lines through transcriptomic profiling. The English language is fine, just need little improvement, some places sentences are not written properly. The manuscript is written properly however I have following comments:

Thank you for your email of June 27, 2022, regarding our manuscript, “Fucoxanthinol Promotes Apoptosis in MCF-7 and MDA-MB-231 Cells by Attenuating Core Signaling Pathways”, and the valuable comments of the reviewers. The changed parts in the manuscript file in accordance with the Reviewer-1’s comments are marked in red that we indicated these pages and lines.

The role of FxOH has been already checked and reported in MCF-7 and MD-MB-231 cell lines, what is the main purpose of the study? Is it just to find out the pathway/s involved in FxOH mediated cytotoxic effect?

We express our thanks for the Reviewer-1’s comments. In accordance with the Reviewer-1’s comments, we have modified title, simple summary, abstract, introduction, and conclusion and emphasized novel findings in the present study. Please confirm “onco-1801563 with Track Change”. (Page 1, line 3, 23-25, 30, and 41-43; Page 3, line 104-105; Page 16, line 438; Page 17, line 552-553).

Line 49: Do the authors mean “BC is the leading cause of cancer death in women”?

Thank you for your constructive comments. Yes. Female BC is the leading causes of cancer deaths for female (Sung et al., 2021): Female mortality, breast 15.5%, lung 13.7%, colorectum 9.5%, cervix uteri 7.7%, stomach 6.0%, liver 5.7%, pancreas 4.9%, ....; Female incidence, breast 24.5%, colorectum 9.4%, lung 8.4%, …..

For avoiding confusing, we have added “Female” in the sentence (Page 2, line 50).

Lines 79-81: Please read the lines and write in proper meaningful way.

Thank you for your valuable comments. In accordance with the Reviewer-1’s comment, we have modified the two sentences (Page 2, line 80-83).

Section 2.2, 2.3, 2.4, 2.5, 2.7 and 2.8: Please mention the exact cell numbers used in each assay, not just the number of cells/ml. Current representation is misleading since the cell numbers change depending on change in the media volume OR provide additional information about the volume of media used for each plate type.

Thank you for your constructive comments. In accordance with the Reviewer’s comments, we have modified the Materials and Method section (2.2, 2.3, 2.4, 2.5, 2.7, amd 2.8). Please confirm the Materials and Method section (Page 3-5, line 136, 144, 153, 164, 200, 228, and 232).

Lines 245-263: Are those observations recorded on Day 2 for both cell type? Please clarify.

Thank you for your constructive comments. In accordance with the Reviewer’s comments, we have modified 3.1. of the Result section (Page 6, line 250, 253, and 256).

Figure 2: Please provide scale bars for Figure 2B.

Thank you for your valuable comments. In accordance with the Reviewer-1’s comment, we have added the scale bars in Figure 2B and the sentence in Figure 2B legends (Page 7, line 278).

Section 3.5: Why authors checked the expression levels of many proteins here when those were not commonly regulated upon FxOH treatment between both the cell lines? Please provide the explanation.

We express our thanks for the Reviewer-1’s comments. In accordance with the Reviewer’s comments, we have modified the sentences (Page 14, line 387-398).

Line 376-379: Sentence is not fully completed, still pending.

Thank you for your valuable comments. In accordance with the Reviewer-1’s comment, we have modified the sentences (Page 14, line 387-398).

Section 3.6: Before stating integrin-β1 or integrin-β4 KD studies, please mention why authors picked them from the proteomic analysis, what is the significance of integrins in the context of cancer progression.

Thank you for your constructive comments. In accordance with the Reviewer-1’s comment, we have added the sentence (Page 16, line 417-420).

Figure 7: Western Blots are not clear; many proteins are not even visible on the blots. Please provide higher exposure for all those proteins which include- Fibronectin, Integrin-β6, Cyclin B1, CCR1, CCR4, CCR7, PRLR, pAKT(Ser⁴⁷³) and both NF-kB blots.

Provide enough β-actin, GAPDH or any loading controls for respective WB panels. Presenting just one loading control for so many blots is not acceptable.

We express our thanks for the Reviewer-1’s comments.

As pointed out by the Reviewer-1, Fibronectin, Integrin-α6, pFAK(Tyr³⁹⁷), Cyclin B1, CCR1, CCR4, CCR7, PRLR, pAKT(Ser⁴⁷³) and NF-kB (p105/p50) could not be visualized. We think the expression and activation levels of these proteins are very low or negative in our experimental condition.

Protein content of each sample was correctly measured by using the Bradford assay in this study. Ten micrograms of sample’s proteins were carefully subjected to SDS-PAGE. Furthermore, we have re-probed the membrane and repeatedly used to determine on the expression and activation of each protein, and finally analyzed β-actin expression. Therefore, we think the assessments of Fibronectin, Integrin-α6, pFAK(Tyr³⁹⁷), Cyclin B1, CCR1, CCR4, CCR7, PRLR, pAKT(Ser⁴⁷³) and NF-kB (p105/p50) are correct. The reliabilities of their antibodies were also previously confirmed.

In accordance with the Reviewer-1’s comments, β-Actin expression of each panel was added in Figure 7. With this modification, the several sentences were modified in the Materials and Methods section (Page 5, line 215-218) and Figure 7 legends (Page 15, line 411-414).

Line 477: Spelling mistake in “induction”. Please correct.

   We apologize for the misrepresentation. We have modified it (Page 17, line 503).

General Comments: Any cytotoxic or cell-static drug will alter multiple pathways which are essentially related to cell cycle, replication, mTOR, MAPK, TGF-β in some context of cancer biology. At the end, authors need to conclude and highlight the precise mechanism (specific pathway/s by which FxOH works). I guess, authors primarily found laminins-integrins axis, if that is the case, please highlight precisely and accordingly the title of the paper may need to be modified.

We express our thanks for the Reviewer-1’s comments. In accordance with the Reviewer-1’s comment, we have modified the title (Page 1, line 3) and Discussion section (Page 16, line 438) and Conclusion section (Page 18, line 552-553).

We feel that the revised manuscript is a suitable response to the comments, and is significantly improved over the initial submission. We trust that is now suitable for publication in Onco.

Thank you in advance for your kind consideration of this paper.

Sincerely yours,

Masaru Terasaki, Ph.D.

Department of Health and Environmental Sciences, School of Pharmaceutical Sciences, Health Sciences University of Hokkaido

1757 Kanazawa, Ishikari-Tobetsu, Hokkaido

061-0293, Japan

Phone: +81-133-23-1211

Fax: +81-133-23-1669

E-mail: [email protected]

Author Response File: Author Response.pdf

Reviewer 2 Report

This manuscript identified the core signaling pathways of Fucoxanthinol-induced anticancer effects on breast cancer cell lines and provides a possible therapeutic agent against breast cancer. However, more detailed description of experimental data and sufficient information is needed before this manuscript can be accepted or publication.

Here are a few additional minor suggestions:

1 In the Figure 2C, Sub-G0/G1content increased post treatment, which indicated that DNA damage causing by FxOH inhibition. If the authors can discuss them in the main text, it would furtherly support their works.

2 The authors hypothesize that FxOH treatment induced apoptosis. If they can provide more evidence about apoptosis as determined by Annexin V/PI staining assay, it would furtherly support their works. 

3 The authors firstly demonstrated that ITGB1 and ITGB4 levels in FxOH-treated MDA-MB-231 breast cancer cells were lower than that in control cells. siRNA-mediated integrin ß1 or integrin ß4 knockdown inhibited cell proliferation. It is better to show the changes of ITGB1 and ITGB4 transcripts via qRT-PCR analysis.

4 24 hours for WST-1 assay may be short.  ITGB1 and ITGB4 depletion may significantly inhibit cell viability at 48 h. It would be better to quantify the cells after a longer incubation to evaluate the difference of cell growth.

Author Response

Reviewer’s comments:

This manuscript identified the core signaling pathways of Fucoxanthinol-induced anticancer effects on breast cancer cell lines and provides a possible therapeutic agent against breast cancer. However, more detailed description of experimental data and sufficient information is needed before this manuscript can be accepted or publication.

Thank you for your email of June 27, 2022, regarding our manuscript, “Fucoxanthinol Promotes Apoptosis in MCF-7 and MDA-MB-231 Cells by Attenuating Core Signaling Pathways”, and the valuable comments of the reviewers. The changed parts in the manuscript file in accordance with the Reviewer-2’s comments are marked in red that we indicated these pages and lines.

Here are a few additional minor suggestions:

1 In the Figure 2C, Sub-G0/G1content increased post treatment, which indicated that DNA damage causing by FxOH inhibition. If the authors can discuss them in the main text, it would furtherly support their works.

We express our thanks for the Reviewer-2’s comments. In accordance with the Reviewer-2’s comments, we have added the sentences in the Discussion section (Page 18, line 509-515).

2 The authors hypothesize that FxOH treatment induced apoptosis. If they can provide more evidence about apoptosis as determined by Annexin V/PI staining assay, it would furtherly support their works. 

Thank you for your valuable comments. In the present study, the enhancement of cellular events containing chromatin condensation, nuclear fragmentation, Sub-G1 ratio, and activation of caspase-3 were observed. Next future, we will investigate the detail mechanisms of the alteration on laminins-integrins axis by FxOH in MCF-7 and MDA-MB-2331 cells. In accordance with the Reviewer-2’s comments, in the next experiment, we would like to try to determine the apoptosis-inducing effects more correctly using Annexin V/PI staining assay. We deeply appreciate your comments.

3 The authors firstly demonstrated that ITGB1 and ITGB4 levels in FxOH-treated MDA-MB-231 breast cancer cells were lower than that in control cells. siRNA-mediated integrin ß1 or integrin ß4 knockdown inhibited cell proliferation. It is better to show the changes of ITGB1 and ITGB4 transcripts via qRT-PCR analysis.

Thank you for your constructive comments. These dsiRNAs are highly reliable products proven in various studies around the world. Therefore, we are strongly confident that the genes of ITGB1 and ITGB4 are undoubtedly downregulated in the MCF-7 and MDA-MB-231 cells.

In the present study, we have investigated the association between the cell growth and integrin β1 or integrin β4 in MCF-7 and MDA-MB-231 cells, using the reliable gene knockdown method. Consequently, significant difference between dsiRNA target and negative control treatments was observed on the cell growth in the both cells in either cases of integrin β1 or β4. First of all, we think that this result is enough for evaluating the association between the cell growth and integrin β1 or integrin β4.

It will take several months to get the result (ITGB1 and ITGB4 transcripts).

However, the Reviewer-2’s comments are very important.

We would like to investigate the changes of ITGB1 and ITGB4 transcripts in the next detail research on FxOH targeting laminins-integrins axis in human BC cells.

4 24 hours for WST-1 assay may be short.  ITGB1 and ITGB4 depletion may significantly inhibit cell viability at 48 h. It would be better to quantify the cells after a longer incubation to evaluate the difference of cell growth.

We express our thanks for the Reviewer-2’s comments. In the gene knockdown experiment, it is of very little importance to make a big difference over a long period of time. It was enough to get significant result on the cell growth by integrin β1 and β4 knockdowns in MCF-7 and MDA-MB-231 cells even for a short time.

Furthermore, their growth was measured at 48 h after from treatments of integrin β1 and β4 dsiRNAs (dsiRNA treatment for 24 h + reinoculation to new plate and incubation for 24 h = total 48 h). In addition, FxOH treatment for 24 and 48 h decreased the cell growths in MDA-MB-231 and MCF-7 cells, respectively. Therefore, we focused on getting the results in a short time incubation, especially to evaluate the effect to the growth by integrin β1 and β4 in MDA-MB-231 cells at 48 hr after from the dsiRNA addition.

Consequently, the both treatments of integrin β1 and β4 dsiRNAs significantly reduced the growth in the both cells (Figure 8).

   We think these data at 24 h is enough for determining the effects of integrin β1 and β4 to the growth in the both cells.

   However, as the Reviewer-2’s comments are very important, we would like to investigate the effects of long integrin β1 and β4 knockdowns to the growth in the both cells in the next research.

We strongly appreciate again for the Reviewer-2’s comments.

We feel that the revised manuscript is a suitable response to the comments, and is significantly improved over the initial submission. We trust that is now suitable for publication in Onco.

Thank you in advance for your kind consideration of this paper.

Sincerely yours,

Masaru Terasaki, Ph.D.

Department of Health and Environmental Sciences, School of Pharmaceutical Sciences, Health Sciences University of Hokkaido

1757 Kanazawa, Ishikari-Tobetsu, Hokkaido

061-0293, Japan

Phone: +81-133-23-1211

Fax: +81-133-23-1669

E-mail: [email protected]

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Authors have addressed all my concerns. I recommend the manuscript in its current version for the publication in the Journal- Onco.