Inhibition of Spontaneous Hepatocarcinogenesis by 4,5-Didehydrogeranylgeranoic Acid: Effects of Small-Dose and Infrequent Administration
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors presented that inhibitory effects of dGGA on spontaneous hepatocarcinogenesis were highly enhanced when a small amount of the compound was timely administered only once or twice. This result was surprising and promising, but a lot of data was lacking to support this conclusion. Why was this drug effective when administered only once or twice? Anti-cancer drug is usually used periodically, such as weekly or once a month. Why was more than 50ug of dGGA less effective? Did some adverse events exist? How did this dGGA prevent cancer development and invasion? The author speculated that GGA induced cell death in cancerous cells, but no results were shown in this manuscript. Some results were presented but the mechanism of action was unclear, and many questions are arising.
Taken together, this manuscript may contain novel results but was not scientific and many data was lacking to support the conclusion.
Author Response
Reviewer 1
The authors presented that inhibitory effects of dGGA on spontaneous hepatocarcinogenesis were highly enhanced when a small amount of the compound was timely administered only once or twice. This result was surprising and promising, but a lot of data was lacking to support this conclusion. Why was this drug effective when administered only once or twice? Anti-cancer drug is usually used periodically, such as weekly or once a month. Why was more than 50ug of dGGA less effective? Did some adverse events exist? How did this dGGA prevent cancer development and invasion? The author speculated that GGA induced cell death in cancerous cells, but no results were shown in this manuscript. Some results were presented but the mechanism of action was unclear, and many questions are arising.
Taken together, this manuscript may contain novel results but was not scientific and many data was lacking to support the conclusion.
We appreciate your insightful comments. We were also surprised when we found the results presented in this manuscript, and we believe they constitute observations of significant clinical importance. Therefore, we will now address each of the questions raised by Reviewer 1 individually.
- Why was this drug effective when administered only once or twice? 　
This is the most significant discovery in this paper. Thank you for your pertinent question. Following general pharmacology principles, we assume that dGGA suppresses carcinogenesis by transmitting its signal through specific receptors or similar pathways in the body, resulting in the biological effects of the drug. Therefore, the question of why the effect can be achieved with one or two administrations translates to what is happening during one or two drug administrations. However, it's important to note that the anti-carcinogenic effect of dGGA is not consistently observed with every one or two administrations. The most notable effect occurs around 11 months of age in C3H mice, prompting the question of what significance this period holds in the spontaneous hepatocarcinogenesis of male C3H mice.
　Several previous papers have reported that in the livers of male C3H mice at around 11 months of age, tumors such as adenocarcinomas are apparently observed macroscopically, with multiple tumors often present within a single liver. It is believed that these tumors serve as precursors that progress into liver cancer a year later. Therefore, administering dGGA at this stage may induce cell death in the tumor cells that were present at this time, effectively eliminating the so-called pre-cancerous cells from the liver at this stage. As a result, we hypothesize that dGGA may inhibit or delay the development of liver cancer one year later (Regarding this point, we have addressed it in the Discussion section of the revised manuscript, spanning from lines 195 to 215 and 278 to 292).
- Why was more than 50ug of dGGA less effective?　
Since the animal experiments spanned a period of two years, we did not conduct a rigorous dose-finding experiment. Therefore, it is currently unclear whether 50 µg of dGGA is the optimal dosage. However, our present study demonstrates that at least 50 µg of dGGA in a single administration was sufficient to observe the anti-carcinogenic effect. While we may have observed the same effect with a single administration of 100 µg or 200 µg, we did not find it necessary to explore these higher doses as we achieved the desired effect with 50 µg. Conversely, lower doses such as 25 µg or 10 µg may also be effective. Indeed, we observed that 3 doses of 5 µg dGGA at 8, 11, and 14 months significantly suppressed carcinogenesis, although less effectively than a single dose of 50 µg dGGA at 11 months, and are now preparing to write a detailed paper on this and other effects of the number and timing of doses on the suppression of carcinogenesis.
　　
- Did some adverse events exist?　
　
First and foremost, dGGA was initially developed as a low-toxicity retinoid. Phase II clinical trials have already been conducted, where individuals were orally administered 600 mg of dGGA daily for one year, with no abnormalities observed in parameters such as blood biochemistry or physical measurements including blood pressure (published in Reference 1 or N. Engl. J. Med. 1996, 334, 1561-1567). In the case of C3H mice, continuous administration for two years, as described in the revised manuscript, did not show any chronic toxicity that could be considered a side effect, including weight loss. Furthermore, acute toxicity from one or two-time administrations was not observed at all. Please see also our response to Reviewer 2's major comment (4).
- How did this dGGA prevent cancer development and invasion?　
The mechanism behind the anti-carcinogenic effect of dGGA on spontaneously occurring liver cancer in C3H mice remains to be elucidated. Moreover, this paper is not focused on dissecting that mechanism, but rather on exploring the feasibility of efficient carcinogenesis inhibition with dGGA. What we can infer about the mechanism from the present experimental results is that a single administration of dGGA around the age of 11 months effectively suppresses the onset of liver cancer. Therefore, it can be hypothesized that the processes occurring around the age of 11 months in the spontaneous development of liver cancer in C3H mice are promptly and irreversibly inhibited by a single administration of dGGA. Considering that dGGA and its endogenous mother compound GGA are known to induce cell death specifically in tumor cells, the most plausible estimation of the anti-carcinogenic mechanism at this stage is that dGGA administered around the age of 11 months efficiently induces cell death in tumor cells involved in the carcinogenesis process and eliminates them from the body, preventing hepatocarcinogenesis.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsIn the current study, Omori et al. studied the potential therapeutic effects of 4,5-Didehydrogeranylgeranoic Acid (dGGA) for hepatocarcinogenesis using C3H/HeNCrj mouse, which is known to develop spontaneous hepatocarcinogenesis. The animal study was conducted with a particular focus on doses and/or timing of dGGA administration. It was demonstrated that lower doses with infrequent administration of dGGA (about 1/3000 to 1/6000 of the total dose used in the study as a high-dose group) showed stronger effects against liver cancer compared to higher doses/longer-term treatment. Although the current study did not approach any potential mechanisms underlying the observed protective effects of dGGA with the used dose(s), the results are intriguing.
There are several issues the authors need to address to improve the manuscript to meet the standards of quality for publication. First, the current manuscript is not well-prepared. Especially, the study method lacks critical information and a part of the discussion is purely hypothetical. Further, the novelty of the current study stands on the lower dose and treatment timing of dGGA. However, the scientific rationality of the choice of dose is not clearly described.
Major comments;
(1) The method section requires more detailed information.
· The authors need to describe how they prepared dGGA solution for oral administration for the animals (What kind of vehicle was used? What volume of solution was given to the animals?) and how they prepared dGGA containing diet.
· The authors need to describe the method(s) by which they performed statistical analysis.
· It is described that "The tumor tissues were processed by routine gross and histological methods with hematoxylin and eosin stained (page 2, lines 79-80)". However, no histological data is shown in the manuscript. The authors need to show corresponding data as figures or remove the corresponding description from the method section.
· It is not clear how they measured the weights of liver tumors of individual animals.
· The authors need to include the housing condition of animals and approval for the animal study from the Institutional Animal Care and Use Committee at the research institute where the study was conducted.
(2) The discussions section requires editing.
The discussion concerning the five points indicated on page 7, lines 256 – 264 should be removed since it is purely hypothetical. The discussion should be mostly to discuss the results from the data shown in the manuscript. Further, some parts seem to be redundant and repetitive. The authors need to make their points more clearly in the discussion.
(3) It is important to clearly describe the scientific rationality of doses used in the current study. In particular, the lower doses of dGCA (i.e., single or dual oral doses with 50 ug/mouse) seem to be a novel treatment strategy for animals. The authors need to explain why the corresponding lower dose and treatment timing(s) were chosen by showing their preliminary data related to dose-response if there are any, or previous publications that describe pharmacokinetics/pharmacodynamics of dGCA in vivo. They mentioned that the relatively small doses of dGCA caused a long-lasting alteration of gene expression, suggesting potential anti-cancer effects with smaller doses of dGCA (page 6, lines 183 – 194). However, the introduced study does not indicate which types of gene expression were changed by the treatment. Further, in the current study, the authors did not examine gene expression in the studied animals. Thus, the point mentioned in the discussion is not sufficient to support the rationality of the treatment design employed in the study.
(4) As basic information, the body weights of studied animals before and after the study need to be shown. It is important information associated with the potential systemic toxicity of dGCA.
Minor comments;
(1) Figure 1. The Y-axis is confusing. It is highly recommended to show the average number of tumors/mouse and the average weight (g) of tumors/mouse separately on the right and left sides of the Y-axis since the two data sets have distinct units (i.e., number and g). In addition, the p values mentioned in the manuscript should be shown in Figure 1.
(2) Table1. The data sources of the average weight of tumors (g/mouse) for the single and dual oral doses are not clear. The values of tumor weights in each condition are average data derived from all different treatment timing (i.e., 2-, 5-, 6-, 8-, 11-, 14-, and 17-months age for single dose, and 22- and 48- weeks of age and 35- and 48- weeks of age for dual doses)?
(3) Although in the result section, the authors use group numbers (i.e., Groups 1-7) to describe studied groups with different conditions, it is not clear which group corresponds to which treatment schedule. In the method section, only Group 2 and Group 4 are described (page 2, lines 74- 75), but other groups are not mentioned.
Comments on the Quality of English LanguageModerate editing of English language required
Author Response
Dear reviewer, please view the attachment
In the current study, Omori et al. studied the potential therapeutic effects of 4,5-Didehydrogeranylgeranoic Acid (dGGA) for hepatocarcinogenesis using C3H/HeNCrj mouse, which is known to develop spontaneous hepatocarcinogenesis. The animal study was conducted with a particular focus on doses and/or timing of dGGA administration. It was demonstrated that lower doses with infrequent administration of dGGA (about 1/3000 to 1/6000 of the total dose used in the study as a high-dose group) showed stronger effects against liver cancer compared to higher doses/longer-term treatment. Although the current study did not approach any potential mechanisms underlying the observed protective effects of dGGA with the used dose(s), the results are intriguing.
We are grateful for your thorough examination and scientifically positive evaluation of our research findings. We are currently in the process of preparing the next report regarding single-dose administration, and the comments from Reviewer 2 are truly encouraging for us.
There are several issues the authors need to address to improve the manuscript to meet the standards of quality for publication. First, the current manuscript is not well-prepared. Especially, the study method lacks critical information and a part of the discussion is purely hypothetical. Further, the novelty of the current study stands on the lower dose and treatment timing of dGGA. However, the scientific rationality of the choice of dose is not clearly described.
Major comments;
(1) The method section requires more detailed information.
- The authors need to describe how they prepared dGGA solution for oral administration for the animals (What kind of vehicle was used? What volume of solution was given to the animals?) and how they prepared dGGA-containing diet.
We added the following sentences to the “Materials and Methods” section.
Using a gastric applicator syringe, 100 μL of cottonseed oil with 50 µg dGGA dissolved in it was orally administered to each subject mouse individually.　　　
Pelleted mouse formula feed was soaked in a solution of dGGA dissolved in ethyl alcohol adjusted to 0.02% of the food for five minutes, then the solution was evaporated completely and dried thoroughly. Eisai Co. (Gifu, Japan) was responsible for the preparation of the diets.
- The authors need to describe the method(s) by which they performed statistical analysis.
We appended the following sentences to the “Materials and Methods” section.
The following methods were used to analyze the statistical data: Number of liver tumors, weight, and tumor-bearing rate: t-test. Test for differences in the distribution pattern of liver tumors: Rigit analysis.
- It is described that "The tumor tissues were processed by routine gross and histological methods with hematoxylin and eosin stained (page 2, lines 79-80)". However, no histological data is shown in the manuscript. The authors need to show corresponding data as figures or remove the corresponding description from the method section.
We appended the following sub-section to the last of the “Results” section.
3.5 The Result of the Histological Examination
The histological examination showed that all the tumors of 100 mg or less were hyper-plastic nodules, and all the tumors of 1000 mg or more were Edmondson typeâ…¡medium-differentiated hepatic microcarcinoma. Details of the results of the histological examination will be shown in our next paper.
- It is not clear how they measured the weights of liver tumors of individual animals.
We added the following sentence to the “Materials and Methods” section.
Liver tumors were weighed individually after carefully removing the tumor portions (separation from the noncancerous portions is relatively easy).
- The authors need to include the housing condition of animals and approval for the animal study from the Institutional Animal Care and Use Committee at the research institute where the study was conducted.
We added the following sentence to the “Materials and Methods” section.
Mice were kept in the animal room of the Gifu University School of Medicine, which is a hygienically controlled facility, after obtaining permission from the Research Ethics Review Committee of the Gifu University School of Medicine.
(2) The discussions section requires editing.
The discussion concerning the five points indicated on page 7, lines 256 – 264 should be removed since it is purely hypothetical. The discussion should be mostly to discuss the results from the data shown in the manuscript. Further, some parts seem to be redundant and repetitive. The authors need to make their points more clearly in the discussion.
According to the reviewer’s suggestion, we removed five points indicated on page 7, lines 256-264, and some other portions were also removed or revised to make our points more clearly.
(3) It is important to clearly describe the scientific rationality of doses used in the current study. In particular, the lower doses of dGCA (i.e., single or dual oral doses with 50 ug/mouse) seem to be a novel treatment strategy for animals. The authors need to explain why the corresponding lower dose and treatment timing(s) were chosen by showing their preliminary data related to dose-response if there are any, or previous publications that describe pharmacokinetics/pharmacodynamics of dGCA in vivo. They mentioned that the relatively small doses of dGCA caused a long-lasting alteration of gene expression, suggesting potential anti-cancer effects with smaller doses of dGCA (page 6, lines 183 – 194). However, the introduced study does not indicate which types of gene expression were changed by the treatment. Further, in the current study, the authors did not examine gene expression in the studied animals. Thus, the point mentioned in the discussion is not sufficient to support the rationality of the treatment design employed in the study.
At the experiment's outset, dGGA was initially considered an acyclic retinoid due to its lack of a cyclic structure. Therefore, the dosage was determined based on the dosage of vitamin supplements. Generally, water-soluble vitamins, which function as coenzymes, exert their effects in the body at the mM order, while fat-soluble vitamins like vitamins D and A, which demonstrate hormone-like actions through nuclear receptors, were believed to exert their effects below the µM order. Since dGGA is an acyclic retinoid, it falls into the category of fat-soluble vitamins. In practice, when using dGGA in a culture system with hepatocellular carcinoma-derived cell lines, it demonstrates differentiation-inducing and cell death-inducing effects at concentrations of 1-10 µM. Therefore, to achieve a concentration of 5 µM with administered dGGA in C3H mice with an average weight of 35g, 53.2 µg of dGGA would be required for systemic diffusion. Based on these considerations, a dosage of 50 µg of dGGA was selected.
As shown in Reference 4, we had already confirmed that a very small dose of retinoids including dGGA induces rapid changes in gene expression and that most of the changes that occur last at least 3 months or longer. It should be noted that Professor Chytil's laboratory had employed 100 µg/animal for rats for years as the dose of vitamin A compound administered to animals to induce physiological changes.
Of course, it is currently unclear whether 50 µg is the optimal dose for male C3H mice. As indicated in our response to Reviewer 1's comment (2), we observed that 3 doses of 5 µg dGGA at 8, 11, and 14 months significantly suppressed carcinogenesis, although less effectively than a single dose of 50 µg dGGA at 11 months, and are now preparing to write a detailed paper on this and other effects of the number and timing of doses on the suppression of carcinogenesis.
(4) As basic information, the body weights of studied animals before and after the study need to be shown. It is important information associated with the potential systemic toxicity of dGCA.
We inserted the additional sub-section to the “Results” section, as shown below.
3.1 Influence of dGGA Administration on the Body Weight of the Mice
The body weight of the mice at the final time point was within the range of 33.0-34.9g in each group including the long–term administration group, and there were no significant differences between groups. dGGA toxicity and side effects have been studied previously, and the doses used in this study, including long-term administration cases, were all less than the toxicity and side effects.
Minor comments;
- Figure 1. The Y-axis is confusing. It is highly recommended to show the average number of tumors/mouse and the average weight (g) of tumors/mouse separately on the right and left sides of the Y-axis since the two data sets have distinct units (i.e., number and g). In addition, the p values mentioned in the manuscript should be shown in Figure 1.
We revised Fig.1 as follows: The average number of tumors/mouse is shown on the leftmost side of Fig.1, and the average weight (g) of the tumor/mouse is shown on the rightmost side, as shown below. Asterisks for p-values are also shown in the figure.
*: p < 0.05, **: p < 0.02, ***: p < 0.01 vs corresponding value of control.
(2) Table 1. The data sources of the average weight of tumors (g/mouse) for the single and dual oral doses are not clear. The values of tumor weights in each condition are average data derived from all different treatment timing (i.e., 2-, 5-, 6-, 8-, 11-, 14-, and 17-months age for single dose, and 22- and 48- weeks of age and 35- and 48- weeks of age for dual doses)?
Thank you for your point. It is true that the explanation regarding the data for all groups was insufficient! We have defined and presented each group in the "Materials and Methods" section as shown below.
50 µg of dGGA was orally administered to the animals only once at different time points, 2 (Group 1), 5 (Group 2), 6 (Group 3), 8 (Group 4), 11 (Group 5), 14 (Group 6), and 17 months (Group 7) after birth, respectively. The control animals (Group 8) were not treated with dGGA during the experimental period.
(3) Although in the result section, the authors use group numbers (i.e., Groups 1-7) to describe studied groups with different conditions, it is not clear which group corresponds to which treatment schedule. In the method section, only Group 2 and Group 4 are described (page 2, lines 74- 75), but other groups are not mentioned.
Thank you for your pertinent remarks; the relationship between the two doses and each group was not fully explained. The detailed explanation is as follows.
Some of the animals in Group 2 of Experiment 2 received a total of 2 dGGA doses at months 5 and 11, which was designated Group 2*, and some in Group 4 received a total of 2 dGGA doses at months 8 and 11, which was designated Group 4*, respectively.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript has been improved and become worth reading.
Author Response
Thank you for your review of our revised manuscript. We especially thank Reviewer 1 for his positive evaluation of our revision. We have responded to Reviewer 2's comments on our revised manuscript point by point as shown below, and we have further revised the manuscript according to those responses. Hence, we are delighted to have the revised manuscript reviewed again.
We are sincerely pleased that thanks to our reviewers' constructive comments we have improved our paper much more than before.
Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors added detailed information in the Material and Methods section, which has improved the manuscript. However, new data and discussion included in the revised manuscript are insufficient to lead readers to their conclusions scientifically. The individual items are listed below. Further, it seems that more detailed data derived from the same cohort study will be included in another manuscript currently prepared. Hence, it is hard to read the current form of the manuscript reporting an important advance that is supported by data that meets the quality standards of publication.
· Although the authors added new section 3.5 providing the histological observations of the tumors found in the studied animals, it is difficult to read the corresponding description as scientific data.
· In the same vein, the way of presenting body weight in the new section 3.1 is not scientific. It is necessary to show mean +/- SD based on the actual body weight of individual animals.
· As mentioned in the comments from reviewers #1 and #2, the dose optimization of dGGA is critical in the current study. Although the authors included related comments in their letter, the revised manuscript does not address this point at all.
· The authors need to make sure the statistical method they employed for the distribution pattern of liver tumors. It is assumed that they performed a Rigid analysis, not a Rigit analysis.
Comments on the Quality of English LanguageModerate editing of English language required
Author Response
Thank you for your review of our revised manuscript. We especially thank Reviewer 1 for his positive evaluation of our revision. We have responded to Reviewer 2's comments on our revised manuscript point by point as shown below, and we have further revised the manuscript according to those responses. Hence, we are delighted to have the revised manuscript reviewed again.
We are sincerely pleased that thanks to our reviewers' constructive comments we have improved our paper much more than before.
Author Response File: Author Response.pdf
Round 3
Reviewer 2 Report
Comments and Suggestions for AuthorsThere are still significant issues that should be addressed before the manuscript is considered for publication. As repeatedly mentioned in the first and second review comments, non-scientific descriptions are still found in the revised manuscript. In addition, the statistical test needs to be reconsidered to have an accurate evaluation of their data as described below.
1. The authors explain in the introduction and discussion sessions that the doses of dGGA used in the current study were determined by the intuition of the authors. First, it is difficult to find scientific soundness in the experiments performed based on intuitive study design. Second, the “intuitive” design of substance administration to animals is not aligned with general IACUC policy. Thus, the authors should rephrase the corresponding context. In addition, if the indicated publication (reference #5) tested dGGA or the comparative compound for the Phase 3 clinical trial, it is highly recommended to convert the dose of dGGA used in the clinical trial to experimental animals by following the previous publication (Nair et al., 2016, J Basic Clin Pharm., PMID: 27057123). In that way, the authors should be able to discuss the doses of dGGA from scientific understanding of dGGA with clinical aspect.
- Discussion: It is described that “However when 50 μg of dGGA is administered to a 35 g mouse and diffused systemically, the concentration is calculated to be approximately 5 μM (page 8, lines 480 – 482)”. Since any reference articles are indicated, this context is assumed to be purely hypothetical. If there is no scientific evidence, the authors should not include this information in the manuscript.
- Figure 1/Table / Table 2: The T-test is not an accurate statistical evaluation for the corresponding data. ANOVA with the Dunnett test needs to be done.
- Results 3.1: The description of body weight. Please explain what * represents for Group 2* and Group 4*.
Extensive editing of English language required
Author Response
please kindly check the attachment
