Background: Although single nucleotide polymorphisms may be potentially important pharmacogenetic determinants of cancer therapy, functional evidence regarding their relevance is currently lacking. The C677T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene is associated with changes in cellular composition of folates. We hypothesized that this polymorphism may modulate the cytotoxic effect of 5-fluorouracil (5FU) and methotrexate (MTX), two commonly used chemotherapeutic agents for colon and breast cancers, because the modes of action of 5FU and MTX are critically dependent on cellular composition of folates. Methods: Human HCT116 colon and MDA-MB-435 breast cancer cells were stably transfected with wild-type or mutant 677T human MTHFR cDNA. MTHFR enzyme activity and thermolability, intracellular folate composition, growth rate, and catalytic thymidylate synthase activity were determined. In vitro chemosensitivity to 5FU and MTX was determined using the sulforhodamine B assay. In vivo chemosensitivity of HCT116 cells to 5FU was determined in nude mice. Results: Compared with cells expressing the wild-type MTHFR, HCT116 and MDA-MB-435 cells expressing the mutant 677T MTHFR had decreased MTHFR activity, MTHFR thermolability, changed intracellular folate distribution, accelerated cellular growth rate, and increased thymidylate synthase activity. The MTHFR 677T mutation increased chemosensitivity of colon and breast cancers to 5FU, but decreased chemosensitivity of breast cancer cells to MTX. In nude mice, xenografts expressing the mutant 677T MTHFR grew faster, but were more sensitive to 5FU, than xenografts expressing the wild-type protein. Conclusions: Our data provide evidence that the MTHFR C677T polymorphism affects the concentration and intracellular distribution of folates and changes the growth and chemosensitivity of colon and breast cancer cells. The MTHFR C677T polymorphism may be a useful pharmacogenetic determinant for providing rational and effective tailored chemotherapy.

Introduction Mankind has been relatively unsuccessful in the search for the ultimate panacea for all ills; however, in the field of functional foods, few nutritional components have so many fundamental and diverse biological properties as folic acid and related B group vitamins. Moreover, few nutrients can claim to modulate, if not overtly benefit, such a wide array of clinical conditions. Around 2500 years ago Hippocrates first espoused the “food as medicine” philosophy, which fell into obscurity by the 19th century. The first 50 years of the 20th century saw the discovery of the essential elements and vitamins, particularly in the context of deficiency diseases. Indeed, by 1912 Casimir Funk had put forward the “vitamine theory,” proposing four different “vitamines” that would cure scurvy, pellagra, beri-beri, and rickets. During the 1970s the shift in emphasis from undernutrition to overnutrition and disease led to a flood of public health guidelines on optimising nutritional parameters. By the 1990s, with an ageing health conscious population, scientists from academia and the commercial world coalesced their thinking to create the trend we now know as functional foods. Enrichment of flour as a US government programme to correct problems with nutrient deficiency was probably the first modern attempt to design a food for functional purposes related to nutritional outcome. The first consequence of this was the eradication of pellagra with niacin, and a current programme in the United States aims to do the same for neural tube defects through mandatory fortification of grain with folate at source.1 2 The ramifications of this mass use of folate as a functional food are likely to have even wider effects. Folate status and common variations in genes that code for folate dependent enzymes are linked to many types of cancer, vascular disease, birth defects, and complications of pregnancy.3 This arises because several molecular mechanisms that underpin the genomic machinery are sensitive to B vitamin status and, in particular, are responsive to the interaction between folate nutrition and folate dependent enzyme polymorphisms (folate nutrigenomics). Mechanisms that may be affected include the maintenance of genomic CpG methylation patterns for regulated gene expression and the proficient synthesis of nucleotides to prevent DNA strand breakage.4 5 The same nutrigenomics also influence plasma homocysteine status and thus risk for vascular disease.3 These relations are shown in figure 1. View larger version: In this window In a new window Fig 1 Molecular mechanisms affected by dietary folate Summary points B vitamins, particularly folate, may give considerable protection against serious diseases such as cancer, heart disease, and birth defects The method of protection is by lowering homocysteine or through epigenetic mechanisms Common single nucleotide polymorphisms of several genes coding for folate dependent enzymes can modulate risk for several serious clinical conditions The level of risk or protection with some single nucleotide polymorphisms is further influenced by a person's nutritional folate status; this is the basis of a new subdiscipline within human nutrition—“nutrigenomics” Folate used in food fortification is not a natural coenzyme; we do not know the long term biological effects of exposure to unmodified synthetic folate Future trends will emphasise dietary practices that complement the genetic variation of an individual person

The impact of folate on health and disease, particularly pregnancy complications and congenital malformations, has been extensively studied. Mandatory folic acid fortification therefore has been implemented in multiple countries, resulting in a reduction in the occurrence of neural tube defects. However, emerging evidence suggests increased folate intake may also be associated with unexpected adverse effects. This literature review focuses on contemporary issues of concern, and possible underlying mechanisms as well as giving consideration the future direction of mandatory folic acid fortification. Folate fortification has been associated with the presence of unmetabolized folic acid (PteGlu) in blood, masking of vitamin B12 deficiency, increased dosage for anti-cancer medication, photo-catalysis of PteGlu leading to potential genotoxicity, and a role in the pathoaetiology of colorectal cancer. Increased folate intake has also been associated with twin birth and insulin resistance in offspring, and altered epigenetic mechanisms of inheritance. Although limited data exists to elucidate potential mechanisms underlying these issues, elevated blood folate level due to the excess use of PteGlu without consideration of an individual's specific phenotypic traits (e.g. genetic background and undiagnosed disease) may be relevant. Additionally, the accumulation of unmetabolized PteGlu may lead to inhibition of dihydrofolate reductase and other enzymes. Concerns notwithstanding, folic acid fortification has achieved enormous advances in public health. It therefore seems prudent to target and carefully monitor high risk groups, and to conduct well focused further research to better understand and to minimize any risk of mandatory folic acid fortification.

To examine the impact of folic acid fortification, including its use as a functional food component, on human health. There is a consensus view that folic acid supplementation has numerous health benefits, many of which are significant in their impact. However, emerging evidence suggests that increased population exposure to folic acid may also have a negative impact with respect to certain developmental and degenerative disorders. As examples, presently much attention is focused on the role of folic acid fortification augmenting colon cancer risk, whereas earlier in the life cycle, the vitamin may additionally influence insulin resistance. Without question, conditions that are influenced by folic acid are both diverse and many - from concerns relating to cognitive decline, breast cancer and vascular disease through to preconceptional issues where maternal folate levels might conceivably alter the phenotype of offspring via epimutations. The highly complex and critical biological importance of folic acid-related molecular nutrition makes it a difficult micronutrient to deploy as a simple intervention at a population level - it has far too many biochemical spheres of influence to predict effects in a generalized way. Additionally, several gene variants and other nutrients are interactive factors. It is, therefore, hardly surprising that the scientific community does not have a true consensus view on whether mandatory fortification is appropriate as a population measure. This latter point not withstanding, any ultimate decisions on fortification should be well rooted in scientific fact rather than political expediency.

A growing number of studies in recent years have highlighted the importance of molecular nutrition as a potential determinant of health and disease. In particular, the ability of micronutrients to regulate the final expression of gene products via modulation of transcription and translation is now being recognised. Modulation of microRNA (miRNA) by nutrients is one pathway by which nutrition may mediate gene expression. miRNA, a class of non-coding RNA, can directly regulate gene expression post-transcriptionally. In addition, miRNA are able to indirectly influence gene expression potential at the transcriptional level via modulation of the function of components of the epigenetic machinery (DNA methylation and histone modifications). These mechanisms interact to form a complex, bi-directional regulatory circuit modulating gene expression. Disease-specific miRNA profiles have been identified in multiple disease states, including those with known dietary risk factors. Therefore, the role that nutritional components, in particular, vitamins and minerals, play in the modulation of miRNA profiles, and consequently health and disease, is increasingly being investigated, and as such is a timely subject for review. The recently posited potential for viable exogenous miRNA to enter human blood circulation from food sources adds another interesting dimension to the potential for dietary miRNA to contribute to gene modulation.