5-10-methylenetetrahydrofolic-acid and Neoplasms

It has been demonstrated that vitamin B6 accumulated in an organism may stimulate the tumour growth as it participates in the formation of polyamines, 5,10-methylene tetrahydrofolic acid and in thymidylate synthetase conformation stabilization. Activation of the tumour growth with the vitamin B6 deficiency is shown to be induced by accumulation of blastomogenic metabolites of tryptophane. The question of vitamin B6 use in oncology remains unsolved.

Topics: Animals; Humans; Neoplasms; Polyamines; Pyridoxine; Tetrahydrofolates; Thymidylate Synthase

Development of tumor-specific probes for imaging by positron emission tomography has broad implications in clinical oncology, such as diagnosis, staging, and monitoring therapeutic responses in patients, as well as in biomedical research. Thymidylate synthase (TSase)-based de novo biosynthesis of DNA is an important target for drug development. Increased DNA replication in proliferating cancerous cells requires TSase activity, which catalyzes the reductive methylation of dUMP to dTMP using (R)-N(5),N(10)-methylene-5,6,7,8-tetrahydrofolate (MTHF) as a cofactor. In principle, radiolabeled MTHF can be used as a substrate for this reaction to identify rapidly dividing cells. In this proof-of-principle study, actively growing (log phase) breast cancer (MCF7, MDA-MB-231, and hTERT-HME1), normal breast (human mammary epithelial and MCF10A), colon cancer (HT-29), and normal colon (FHC) cells were incubated with [(14)C]MTHF in culture medium from 30 min to 2 h, and uptake of radiotracer was measured. Cancerous cell lines incorporated significantly more radioactivity than their normal counterparts. The uptake of radioactively labeled MTHF depended upon a combination of cell doubling time, folate receptor status, S phase percentage, and TSase expression in the cells. These findings suggest that the recently synthesized [(11)C]MTHF may serve as a new positron emission tomography tracer for cancer imaging.

Topics: Cell Division; Cell Line, Tumor; Deoxyuracil Nucleotides; DNA Replication; DNA, Neoplasm; Humans; Methylation; Neoplasm Proteins; Neoplasms; Positron-Emission Tomography; Radioactive Tracers; Radiography; Tetrahydrofolates; Thymidine Monophosphate; Thymidylate Synthase

5-fluorouracil forms classic (covalent, ternary) complexes consisting of thymidylate synthase, fluoro-deoxyuridine monophosphate, and 5,10-methylene tetrahydrofolate. Despite a high pharmacologic interest in the classic complexes formed in cells treated with fluorouracil anticancer agents, the in vivo stability of the complexes and the possible interference in complex formation by other coadministered compounds have not been adequately described. We visualized classic complexes unaccompanied by unbound thymidylate synthase, inferring complete enzymatic inhibition, in 5-fluorouracil-treated S. cerevisiae and cancer cells in vitro and in murine tumors in vivo treated with 5-fluorouracil. Classic complexes persisted 13 days in cancer cells after a pulse of 5-fluorouracil. Classic complexes were reduced to absent in cancer cells in which the older antifolates methotrexate and aminopterin, or the modern antifolates pemetrexed and tomudex, were coadministered with 5-fluorouracil. Classic complexes were, however, detected when an alternate drug, 5-fluorodeoxyuridine, was administered with methotrexate. We visualized classic complexes at fifteen minutes to seven days after an acute single dose of 5-fluorouracil in mouse tumor models, in tumors and normal tissues. Using the same assay, we detected unbound thymidylate synthase in untreated human tissues, supporting the future use of this assay in evaluating the most appropriate dose of fluoropyrimidine and coadministered agents in clinical settings.

Topics: Animals; Female; Fluorouracil; Folic Acid Antagonists; Humans; Methotrexate; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasms; Saccharomyces cerevisiae; Tetrahydrofolates; Thymidylate Synthase; Time Factors; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Topics: Chromatography, High Pressure Liquid; Drug Monitoring; Folic Acid Antagonists; Humans; Leucovorin; Methotrexate; Neoplasms; Tetrahydrofolates

Serine hydroxymethyltransferase (SHMT) is a ubiquitous enzyme found in all prokaryotes and eukaryotes. As an enzyme of the thymidylate synthase metabolic cycle, SHMT catalyses the retro-aldol cleavage of serine to glycine, with the resulting hydroxymethyl group being transferred to tetrahydrofolate to form 5, 10-methylene-tetrahydrofolate. The latter is the major source of one-carbon units in metabolism. Elevated SHMT activity has been shown to be coupled to the increased demand for DNA synthesis in rapidly proliferating cells, particularly tumour cells. Consequently, the central role of SHMT in nucleotide biosynthesis makes it an attractive target for cancer chemotherapy.. We have solved the crystal structure of human cytosolic SHMT by multiple isomorphous replacement to 2.65 A resolution. The monomer has a fold typical for alpha class pyridoxal 5'-phosphate (PLP) dependent enzymes. The tetramer association is best described as a 'dimer of dimers' where residues from both subunits of one 'tight' dimer contribute to the active site.. The crystal structure shows the evolutionary relationship between SHMT and other alpha class PLP-dependent enzymes, as the fold is highly conserved. Many of the results of site-directed mutagenesis studies can easily be rationalised or re-interpreted in light of the structure presented here. For example, His 151 is not the catalytic base, contrary to the findings of others. A mechanism for the cleavage of serine to glycine and formaldehyde is proposed.

Topics: Binding Sites; Catalytic Domain; Crystallography, X-Ray; Cytosol; Dimerization; DNA Replication; Drug Design; Enzyme Inhibitors; Glycine; Glycine Hydroxymethyltransferase; Humans; Models, Chemical; Models, Molecular; Molecular Sequence Data; Neoplasms; Protein Conformation; Protein Folding; Recombinant Proteins; Serine; Tetrahydrofolates

This report details our methods for performance of the major parameters related to quantitation of TS inhibition resulting from fluoropyrimidine administration to patients, methods equally applicable to preclinical studies. Sampling of tumors before and after drug treatment is done by 4 mm disposable punch biopsy or forceps biopsy via subcutaneous tunneling. Homogenates are prepared using N2 or polytron-mincing. Cytosolic free TS is measured by either the tritium-release method for small biopsies or by [3H]FdUMP ligand-binding. FdUMP and dUMP are separated by DEAE-cellulose column and measured by competitive binding and [14C]dTMP synthesis by the Moran methods. Total, post-FUra TS is measured by pre-incubation dissociation of FdUMP-bound TS after neutral charcoal removal of cytosolic ligands. H4PteGlu and CH2-H4PteGlu are measured by the Priest method using L. Casei TS. The materials and methods are described in sufficient detail to permit wide application of this approach.

Topics: Biopsy, Needle; Cytosol; Deoxyuracil Nucleotides; Fluorodeoxyuridylate; Humans; Lacticaseibacillus casei; Neoplasms; Radioisotope Dilution Technique; Tetrahydrofolates; Thymidylate Synthase; Tritium