Induction of Hepatoma Cell Pyroptosis by Endogenous Lipid Geranylgeranoic Acid—A Comparison with Palmitic Acid and Retinoic Acid

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors Yoshihiro Shidoji

• PhD
• Principal Investigator at University of Nagasaki

Japan   The manuscript relates to the current state of anti-Oncometabolites and their application to cancer cells and as cancer therapeutics. Dr. Shidoji is the leading expert in this field. A search in PubMed https://pubmed.ncbi.nlm.nih.gov/ demonstrates that there are over 50 publications describing aspects of GGA. More than 50% of them include Dr. Shidoji as a main author.  Similar situation regarding GGA and cancer. GGA reviews account for 5 publications, three of them are authored by Dr. Shidoji. A recent 2023 publication by Dr. Shidoji explores more of the chemistry aspects of GGA as a bioactive metabolite.   The manuscript does not require further evaluation and should be accepted as is after a review by the author that nothing is missing. The tables are extensive and full of details and citations. That makes it almost impossible to confirm all of the information in 1 week.   Missing from this manuscript is a more detailed discussion regarding GGA's partners, like TLR4. Personally, I would have liked to see more of the mechanism of action. Why is TLR4 considered but discouraged as the sole responsible for driving GGA's signaling/effects? Do these hydrophobic compounds, like GGA, interact directly with the membrane? Do they move into the cell after melting through the plasma membrane like steroids and cholesterol? If these are interesting questions, then what is preventing the field from answering these questions? Is a synthesis problem, a model's problem, etc. As an expert in this molecule since 1995, it will be good to hear from you how you see the research progress. What are the difficulties and bottlenecks of the research? Why it has not been explored further in vivo? Some of the effects of GGA and other anti-oncometabolite have been demonstrated for over two decades. Are there any clinical studies that have been completed or underway? The manuscript only mentions Muto et al 1996. That is almost 30 years ago. It would be very good to challenge the current state of the art of clinical studies of anti-oncometabolite.            

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The review by Shidoji et al., summarises the latest finding on GGA-mediated pyroptotic cell death and comprehensively compares with the cell death induced by palmitic acid and retinoic acid.

 

The review is very condense and difficult to follow at times. The review is more focused on technical details rather than providing conceptual knowledge. For example, 2.1 where timeline has been described can be summarized in a table or a schematic diagram. A detailed explanation that occur  with GGA treatment seem unnecessary and overwhelming.

 

Similarly, too much details on the concentrations and timing of various treatment. In general reviews should focus on the implications and unique properties rather than technical details.

 

Throughout the manuscript the authors jump from one mechanism to the other without giving a context. Can the authors summarize or focus on whats similar and different between GGA, ATRA and PA (not focusing on the concentrations or timing of treatment).

 

GGA inducing pyroptosis should be elaborated and the distinction from PA should be clarified elegantly. As such, it is very confusing whats the author is trying to convey as too many technical details act as a distraction (increasing the content of writing) rather than informative.

 

If GGA works through TLR4, how GGA selectively kills the cancer cells but not normal hepatocytes. This should be discussed.

 

Can the authors make a schematic diagram detailing the difference between GGA, PA and ATRA (as in figure 1). Please refrain from using doses and timing details, if such a schematic diagram is provided.

 

Comments on the Quality of English Language

None

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

1.     The author compares the mechanism of HCC inhibition by Geranylgeranoic Acid with Palmitic Acid and Retinoic Acid, based on intracellular events occurring in HCC cells after GGA treatment, as reported by different studies and they discuss the inhibition of GGA-induced cell death by various inhibitors. The timeline of intracellular events, thus created, may not be real as these reports use different doses of GGA and varying experimental settings.

 

2.     The author summarizes the inhibition of GGA-induced cell death by various inhibitors, which indicates towards multiple pathways that drive the anti-oncogenic action of GGA.

 

3.     Most of the studies cited by the author are based on in vitro data from cell lines, particularly hepatocytes. However, the title is too generalized to reflect the same.

 

4.     HCC demonstrates an escalated high rate of glycolysis. How does GGA affect glycolysis in HCC cells?

 

5.     Section 3.4, line 424-425, the author states that ‘the effects of the three fatty acids on human HCC cells can be broadly categorized into two types: pyroptosis induced by GGA and PA, and apoptosis 425induced by ATRA’. However, several studies have reported the induction of apoptosis by PA in hepatocytes.

6.     The authors need to add if there are any specific pathways or receptors targeted by geranylgeranoic acid that are distinct from those targeted by palmitic acid and retinoic acid

7.     Please add if any synergistic effects observed when combining geranylgeranoic acid with palmitic acid or retinoic acid in inhibiting hepatocellular carcinoma growth?

 

8.     Please add the effects of these endogenous lipids on the tumor microenvironment, including immune cell infiltration, cytokine secretion, and angiogenesis, and how these effects influence hepatocellular carcinoma progression?

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed my comments. Especially, I liked the timeline representing various cellular events.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript has improved.