From Fly to Human: Translational Relevance of Drosophila Models in the Study of Vitamin B6 and Cancer Relationship

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript provides a high-quality, comprehensive, and mechanistically insightful review of the complex relationship between vitamin B6 metabolism and cancer, highlighting the powerful translational relevance of Drosophila melanogaster models.

Major Strengths

1. Strong Conceptual Integration
   The review elegantly connects biochemical pathways (PLP metabolism, one-carbon metabolism), genomic instability, oxidative stress, immune modulation, and cancer progression into a coherent mechanistic framework.

2. Innovative Translational Angle
   The emphasis on Drosophila as a tool to dissect gene–nutrient interactions and metabolic vulnerabilities is original, compelling, and highly convincing, offering perspectives not achievable through epidemiology or cell culture alone.

3. Mechanistic Depth
   The sections on:

   • SHMT–PLP interaction,

   • ROS-mediated genomic instability,

   • LOH induction, and

   • Diabetes-cancer connections

   provide exceptional mechanistic clarity and translational relevance.

4. Clinical Relevance
   The discussion of PLP levels, PNPO/PDXK dysregulation, and metabolic vulnerabilities of cancer cells is highly relevant for precision oncology and metabolic-based therapeutic strategies.

Minor Suggestions for Further Improvement

These suggestions are optional and aimed solely at enhancing clarity and impact, not at correcting major weaknesses.

1. Add a Short Clinical Perspective Box
   Consider adding a short boxed section discussing:

   • Potential implications for biomarker development,

   • Nutritional interventions,

   • Combination strategies involving vitamin B6 metabolism and chemotherapy.

2. Expand Discussion on Therapeutic Risks
   Since PLP depletion may induce genomic instability, a short paragraph highlighting possible risks of indiscriminate PLP inhibition in cancer therapy would provide a valuable translational caution.

3. Clarify Context-Dependency More Explicitly
   While well discussed, a concise summary figure or table highlighting:

   • Protective vs tumor-promoting roles of vitamin B6
     across different cancer stages and types could further improve clarity.

Overall Evaluation for Authors

This manuscript represents a high-impact, authoritative, and conceptually innovative review that will be of significant interest to researchers in cancer metabolism, nutritional oncology, genomic instability, and model organism biology. The work is highly suitable for publication in IJMS after only minor optional refinements.

Author Response

1. Add a Short Clinical Perspective Box
   Consider adding a short, boxed section discussing:
• Potential implications for biomarker development,
• Nutritional interventions,
• Combination strategies involving vitamin B6 metabolism and chemotherapy.
2. Expand Discussion on Therapeutic Risks
   Since PLP depletion may induce genomic instability, a short paragraph highlighting possible risks of indiscriminate PLP inhibition in cancer therapy would provide a valuable translational caution.
3. Clarify Context-Dependency More Explicitly
   While well discussed, a concise summary figure or table highlighting:
• Protective vs tumor-promoting roles of vitamin B6
  across different cancer stages and types could further improve clarity.

 

We would like to thank the reviewer for the constructive comments. We have addressed all the points raised and believe that the manuscript has been significantly improved as a result. In particular,

1. We have added a short Clinical Perspective box, as requested (after the Conclusions section).
2. We have expanded the Discussion section regarding the effects of PLP depletion in healthy cells in the context of cancer therapy.
3. We have added Figure 5 to better clarify the role of PLP in different cellular contexts.

 

Reviewer 2 Report

Comments and Suggestions for Authors

Researchers from Vernì et al. published this review article to highlight the twofold role of vitamin B6 and its active form PLP in relation to cancer prevention and therapy. Vitamin B6 deficiency leads to genotoxic stress by increasing the levels of ROS and also by impairing the single carbon metabolism in which it plays a central role. According to the authors Drosophila models are an excellent biological tool to understand and clarify the gene–nutrient interactions that explain the role of vitamin B6/SHMT1 pathway in cancer. Additionally, the authors suggest a link between the increased cancer risk of diabetic patients with vitamin B6 deficiency by LOH. All in all, this paper establishes a molecular biological foundation for the precision therapy of cancer based on the understanding of nutrient metabolism.

 

On my opinion, the following are the specific contributions of this paper to academic research:

1. Addressing the limitations of human research and the dilemma of causality.

This review focuses mainly on how research with the Drosophila in a highly controlled environment can eliminate a multiplicity of confounding variables in experimental biology that lead to a direct demonstration of the aetiological relationship between vitamin B6 deficiency and cancer.

2. Revealing the core mechanism of ROS-driven malignant transformation.

Original Research Paper Important academic value molecular model: Vitamin B6  deficiency leads to the increase of ROS through two ways.

3. Discovering a new trigger for LOH.

An original finding of this paper is that B6 deficiency leads to LOH.

4. Establishing the therapeutic value of gene-nutrient interactions.

Pre-clinical studies suggest that compounds targeting PLP-dependent enzymes can induce apoptosis and inhibit the growth of tumor cells overexpressing SHMT and produce a synergistic anti-tumor effect, supporting the concept of metabolism-based precision medicine.

5. Elucidating the mechanistic link between diabetes and cancer risk.

The article observed that in hyperglycemia condition, a vitamin B6 deficiency will accelerate the formation of AGEs. Such AGEs can enhance oxidative stress and DNA injury. The article believes that the vitamin B6 deficiency can serve as a mechanistic link between diabetes and cancer.

This article contributes to the filed of clinical pathology by summing up scattered clinical phenomena into a molecular pathologic mechanism. Vitamin B6 is proved to be the first-line genomic guardian and also provided an innovative idea for cancer prevention and therapy.

 

This review article, as well as current research on vitamin B6 and cancer, has the following main shortcomings and limitations.

1. Inconsistencies in Human Research Data

The analysis of the available data revealed a strong and highly consistent negative correlation between plasma PLP and cancer risk, whereas the analysis of dietary intake led to very inconsistent conclusions.

Preventive effect in well-nourished population uncertain: More calcium may not provide additional benefit.

Although previous research indicated that B6 in dietary supplements is beneficial for health, a new study suggests that B6 found in food alone is not as health-promoting. The researchers say that B6 in the diet is generally part of a complete system that confers benefit, but that the overall amount of B6 from both diet and supplements has not been shown to support health.

2. Confounding Factors in Epidemiological Studies

Establishing clear causal relationships is difficult in human studies.

Health behavior interference: Most studies measure vitamin intake in individuals who generally have other healthy habits as well – such as exercising and not smoking. This can make it difficult for researchers to isolate the role of vitamin B6.

In the complex mixture of the diet the interactions between nutrients make it very difficult to assess which effect of vitamin B6 is primary and which is secondary. We almost always consume nutrients with other nutrients, so any apparent effect of vitamin B6 can also be due to many other factors present in the food.

3. Molecular Mechanisms Not Fully Elucidated

Vitamin B6 is associated with diabetes, cancers and neurodegenerative diseases. The roles of vitamin B6 in these diseases are not yet fully understood and the PLP catabolism in humans and mammals generally remain a mystery.

4. Differences in Mechanisms Among Species

Fruit flies are a common lab model because they have shorter lifespans and can be made to carry mutations that don’t always occur in more complex species like mammals and humans. But they are not the only model in which DNA damage has been studied. For example, researchers found that DNA damage in yeast caused by a B6 deficiency was a result of the imbalance in nucleotides, a result that differed from the uracil intrusion previously reported in fruit flies, suggesting that the results are not directly applicable to humans.

5. Challenges in Clinical Translation

Therapeutic Toxicity: Although early work suggested considerable potential for compounds like 4-DP as anticancer drugs, their clinical applicability has been precluded by adverse effects when given systemically.

One of the challenges of precision medicine is that the involvement of PLP in global metabolism means that any treatment designed to modulate PLP activity must be delivered with extraordinary precision to have any therapeutic effect.

6. Insufficient Sample Size in Specific Studies

There is very limited data on gene-nutrient interactions from human clinical trials. For example, as mentioned previously, the SHMT1 genotype polymorphism and its interaction with vitamin B6 and risk of breast cancer has been investigated in a very small clinical trial of 131 women. A larger human clinical trial with more participants would be required to confirm these findings.

Some concerned issues were raised:

1. Inconsistency between "Intake" and "Plasma Concentration"

Plasma PLP levels have been reported to be negatively correlated with a risk of cancer; however, the association between dietary intake and cancer risk has been the subject of variable reports.

If PLP is required for genome stability how can intakes be increased without failure to do so resulting in a reduction in the risk of cancer? Is the heterogeneity of bioavailability of nutrients in the diet sufficient to explain the failure of increasing the dietary levels of nutrients or their analogues to result in an aetiological reduction in cancer? Or is the physiological level for such required nutrients sufficiently high to prevent a beneficial effect of increasing their intake, and consequently would increasing intakes merely be futile? Given the significance of diet in the aetiology of this and other disease processes it is likely that prevention of cancer will not be solely diet related.

2. The Dilemma of "B6 Supplementation" and "Cancer Addiction"

According to the source, B6 deficiency can lead to cancer. Meanwhile, existing cancers (such as some forms of AML or ovarian cancer) can become dependent on B6 for their growth.

Points to Consider • When to Vitamin B6 Supplment and when to Vitamin B6 Restrict • How to Distinguish Precancerous Cells and Healthy Cells If we suspect that a patient has precancerous cells, and healthy cells, when shall we give Vitamin B6 to prevent gene mutations, and when shall we restrict Vitamin B6 so as not to let the growing cancer cells get too much of it. In the case when Vitamin B6 protects normal cells while not benefiting the pre-cancerous cells, will it not rather speed up the emergence of cancer.

3. The Gap Between the Drosophila Model and Human Complexity

“Despite great molecular conservation in Drosophila, we acknowledge that there might be a difference between species such as yeast and fruit flies.”

Points to consider: - Neuroblasts in the brain of a Drosophila larva are rapidly dividing cells. Is this comparable to a broad class of human cancers such as those found in slow growing organs such as prostate and breast? - Surprisingly little is known concerning the metabolism of B6 in man, which may be an additional complicating factor in trying to relate the findings with fruit flies to potential new treatments for man.

4. Practicality of Precision Medicine

The authors then suggest that using a PLP inhibitor to target tumors that overexpress SHMT would be a good direction. They do point out that PLP is involved in only 4% of global systemic metabolism; and that when previous inhibitors were tried, they were so toxic in vivo that they weren’t tolerable.

Is there an inhibitor of pyridoxine-5-phosphate phosphatase available that can specifically inhibit B6 metabolism at the tumor site, without toxic effects on the brain or on the immune system? Is “precision medicine” naively unrealistic in respect to essential nutrients like B6, which is involved in countless biological processes?

5. The Isolation of Nutrient Interactions

Based on the source data, nutrients are assumed to work synergistically. For example, B6, B9 and B12 are required for single-carbon metabolism. In fact, the protective role of vitamin B6 is closely related to the level of folate. In addition, it is doubtful whether the study of the carcinogenic threshold of vitamin B6 in separate studies is of practical significance to clinical prevention. Perhaps we should even consider establishing an index of “metabolome” for clinical prevention, instead of evaluating the concentration of a single vitamin.

6. In terms of diabetes, what is the causal direction?

A study indicates that diabetic patients typically demonstrate low levels of vitamin B6 and that the deficiency can increase the amount of damage to DNA by diabetes.Is there an issue of causation? Is there any possibility that patients with diabetes deplete their vitamin B6 stores quicker than those without or have patients with a long-standing deficiency in vitamin B6 developed diabetes? It is critical to ascertain the answer to these questions for any future possible intervention(s)

Author Response

1. Inconsistency between "Intake" and "Plasma Concentration"

Plasma PLP levels have been reported to be negatively correlated with a risk of cancer; however, the association between dietary intake and cancer risk has been the subject of variable reports.

If PLP is required for genome stability how can intakes be increased without failure to do so resulting in a reduction in the risk of cancer? Is the heterogeneity of bioavailability of nutrients in the diet sufficient to explain the failure of increasing the dietary levels of nutrients or their analogues to result in an aetiological reduction in cancer? Or is the physiological level for such required nutrients sufficiently high to prevent a beneficial effect of increasing their intake, and consequently would increasing intakes merely be futile? Given the significance of diet in the aetiology of this and other disease processes it is likely that prevention of cancer will not be solely diet related.

We thank the reviewer for all his/her comments

We agree that multiple explanations may account for the lack of a clear protective effect of increased PLP intake, including differences in bioavailability and the possibility that physiological levels are already sufficient. However, we would also like to highlight that limitations inherent to epidemiological studies may contribute to this observation. In particular, measurement error in dietary assessment, inter-individual variability in nutrient absorption and metabolism, residual confounding, and potential threshold effects may obscure true associations. Therefore, the absence of a consistent epidemiological signal should not be interpreted as definitive evidence of a lack of a biological effect.

1. The Dilemma of "B6 Supplementation" and "Cancer Addiction"

According to the source, B6 deficiency can lead to cancer. Meanwhile, existing cancers (such as some forms of AML or ovarian cancer) can become dependent on B6 for their growth.

Points to Consider • When to Vitamin B6 Supplement and when to Vitamin B6 Restrict • How to Distinguish Precancerous Cells and Healthy Cells If we suspect that a patient has precancerous cells, and healthy cells, when shall we give Vitamin B6 to prevent gene mutations, and when shall we restrict Vitamin B6 so as not to let the growing cancer cells get too much of it. In the case when Vitamin B6 protects normal cells while not benefiting the pre-cancerous cells, will it not rather speed up the emergence of cancer.

We thank the reviewer for raising this important point regarding the dual role of vitamin B6 (pyridoxal 5′-phosphate, PLP) in cancer biology. While PLP deficiency may contribute to genomic instability and increase cancer risk, some established tumors, including certain forms of AML and ovarian cancer, may depend on PLP for their growth.

Supplementation may be most relevant under conditions of nutritional deficiency or when intracellular PLP levels are low, thereby protecting genome stability in normal cells. It is unlikely that B6 supplementation in normal cells would trigger cancer, which is a multistep and multifactorial process.

Conversely, in early-stage tumors, modest reductions in PLP availability could, in principle, preferentially affect tumor cells (which may be more sensitive due to increased PLP utilization) while minimally impacting healthy cells. In late-stage tumors, where B6 is progressively depleted, supplementation could help prevent further cancer progression.

We emphasize that these considerations are speculative and intended to highlight mechanistic possibilities rather than provide clinical recommendations.

3. The Gap Between the Drosophila Model and Human Complexity

“Despite great molecular conservation in Drosophila, we acknowledge that there might be a difference between species such as yeast and fruit flies.”

Points to consider: - Neuroblasts in the brain of a Drosophila larva are rapidly dividing cells. Is this comparable to a broad class of human cancers such as those found in slow growing organs such as prostate and breast? - Surprisingly little is known concerning the metabolism of B6 in man, which may be an additional complicating factor in trying to relate the findings with fruit flies to potential new treatments for man.

While it is true that Drosophila neuroblasts are rapidly dividing cells and may not fully recapitulate the growth dynamics of slower-proliferating human tumors such as those of the prostate or breast, Drosophila remains a valuable model for studying fundamental mechanisms of tumorigenesis. Notably, the best-characterized Drosophila tumor models are epithelial, like the majority of human cancers, and many of the genes and signaling pathways involved in tumor development are highly conserved. This conservation allows mechanistic insights gained in flies to increase our understanding of genome maintenance, metabolic regulation, and tumor biology in higher organisms.

We also acknowledge that the metabolism of vitamin B6 in humans is still not fully understood, which complicates the extrapolation of findings from Drosophila directly to potential therapeutic applications. Nevertheless, studies in Drosophila provide a tractable system to uncover conserved cellular and molecular principles that may guide further investigations in mammalian systems.

 

1. Practicality of Precision Medicine

 

The authors then suggest that using a PLP inhibitor to target tumors that overexpress SHMT would be a good direction. They do point out that PLP is involved in only 4% of global systemic metabolism; and that when previous inhibitors were tried, they were so toxic in vivo that they weren’t tolerable.

Is there an inhibitor of pyridoxine-5-phosphate phosphatase available that can specifically inhibit B6 metabolism at the tumor site, without toxic effects on the brain or on the immune system? Is “precision medicine” naively unrealistic in respect to essential nutrients like B6, which is involved in countless biological processes?

We thank the reviewer for raising these important points regarding the feasibility and safety of targeting PLP in tumors. We have addressed these considerations in the revised Discussion section. We clarified that, given the essential roles of PLP in global metabolism and genome maintenance, any strategy involving PLP depletion would require a targeted precision-medicine approach to minimize systemic toxicity and mutagenic effects in normal cells. Approaches such as tumor-specific delivery of siRNA against genes involved in vitamin B6 metabolism, such as PDXK, may help limit off-target effects. We also speculate that tumor cells more dependent on PLP may be particularly vulnerable; in such contexts, even modest reductions in PLP could significantly impact cancer growth while sparing normal tissues. Importantly, these considerations highlight the conceptual rationale while acknowledging the substantial challenges in translating PLP-targeted strategies into safe therapeutic interventions.

1. The Isolation of Nutrient Interactions

Based on the source data, nutrients are assumed to work synergistically. For example, B6, B9 and B12 are required for single-carbon metabolism. In fact, the protective role of vitamin B6 is closely related to the level of folate. In addition, it is doubtful whether the study of the carcinogenic threshold of vitamin B6 in separate studies is of practical significance to clinical prevention. Perhaps we should even consider establishing an index of “metabolome” for clinical prevention, instead of evaluating the concentration of a single vitamin.

We thank the reviewer for this insightful comment. We agree that nutrients often act synergistically and that the protective role of vitamin B6 is closely linked with folate (B9) and vitamin B12. The reviewer’s suggestion of considering a broader metabolomic approach for clinical prevention is indeed interesting and may represent a valuable direction for future research.

 

6. In terms of diabetes, what is the causal direction?

A study indicates that diabetic patients typically demonstrate low levels of vitamin B6 and that the deficiency can increase the amount of damage to DNA by diabetes. Is there an issue of causation? Is there any possibility that patients with diabetes deplete their vitamin B6 stores quicker than those without or have patients with a long-standing deficiency in vitamin B6 developed diabetes? It is critical to ascertain the answer to these questions for any future possible intervention(s).

We thank the reviewer for raising this important point regarding causality. Low vitamin B6 levels in diabetes may be both a cause and a consequence of the disease (see ref.#20). On one hand, B6 deficiency can contribute to oxidative stress, DNA damage, and metabolic dysregulation, potentially promoting the development or progression of diabetes. On the other hand, diabetes itself may deplete PLP through increased protein metabolism—raising enzymatic demand—and through chronic inflammation, which mobilizes PLP to inflamed tissues. Regardless of causality, these observations suggest that restoring PLP levels through supplementation could be beneficial, either to prevent further DNA damage in diabetic patients or to mitigate complications associated with low PLP. We agree that clarifying the directionality of this relationship is critical for the design of future intervention studies

Reviewer 3 Report

Comments and Suggestions for Authors

Title: From fly to human: translational relevance of Drosophila models in the study of vitamin B6 and cancer relationship

Comments:

In this manuscript (review), the authors showed the utility of Drosophila melanogaster as a model to elucidate the molecular pathways linking vitamin B6 deficiency to a range of malignant tumoral lesions. Since experimental in vivo protocols in humans and laboratory animals have several limitations, alternative models of cancer occurrence and evolution are essential. Overall, the authors pimpont the major advantages of Drosophila model in vitamin B6 and cancer research such as:  (1) a highly controlled environment, (2) the model facilitates the establishment of a clear cause-and-effect relationships, (3) the model enables a detailed exploration of underlying molecular mechanisms, (4) it facilitates an ease with which exogenous compounds can be administered and metabolized, (5) the Drosophila model exhibits fewer toxic side-effects when treated with PLP inhibitors, and (6) its relatively simple karyotype allows a precise analysis of chromosomal damage. Concerning vitamin B1, along with its active form, pyridoxal 5′-8 phosphate (PLP), by utilizing several retrospective and prospective reports, the authors present the actual situation concerning the correlation between dietary vitamin B6 intake (and plasmatic PLP levels) and cancer risk. Accordingly, PLP deficiency perturbs genomic stability, acting as a driver of oncogenesis in non-transformed cells, whereas in established malignancies the response is highly heterogeneous. Furthermore, studies on flies suggested that PLP deficiency may exacerbate cancer risk in diabetic patients, whereas B6 deficiency can trigger tumorigenesis through loss of heterozygosity (LOH). In view of this, the presented review is interesting and may have great importance for human (and veterinary) oncology. Overall, the paper is well-documented and written, and the results are clearly presented.

Author Response

We sincerely thank the reviewer for their careful assessment of our manuscript and for their positive and supportive comments.