Folic acid mediates activation of the pro-oncogene STAT3 via the Folate Receptor alpha.

The signal transducer and activator of transcription 3 (STAT3) is a well-described pro-oncogene found constitutively activated in several cancer types. Folates are B vitamins that, when taken up by cells through the Reduced Folate Carrier (RFC), are essential for normal cell growth and replication. Many cancer cells overexpress a glycophosphatidylinositol (GPI)-anchored Folate Receptor α (FRα). The function of FRα in cancer cells is still poorly described, and it has been suggested that transport of folate is not its primary function in these cells. We show here that folic acid and folinic acid can activate STAT3 through FRα in a Janus Kinase (JAK)-dependent manner, and we demonstrate that gp130 functions as a transducing receptor for this signalling. Moreover, folic acid can promote dose dependent cell proliferation in FRα-positive HeLa cells, but not in FRα-negative HEK293 cells. After folic acid treatment of HeLa cells, up-regulation of the STAT3 responsive genes Cyclin A2 and Vascular Endothelial Growth Factor (VEGF) were verified by qRT-PCR. The identification of this FRα-STAT3 signal transduction pathway activated by folic and folinic acid contributes to the understanding of the involvement of folic acid in preventing neural tube defects as well as in tumour growth. Previously, the role of folates in these diseases has been attributed to their roles as one-carbon unit donors following endocytosis into the cell. Our finding that folic acid can activate STAT3 via FRα adds complexity to the established roles of B9 vitamins in cancer and neural tube defects.

Absorption and intermediary metabolism of purines and pyrimidines in lactating dairy cows.

About 20 % of ruminal microbial N in dairy cows derives from purines and pyrimidines; however, their intermediary metabolism and contribution to the overall N metabolism has sparsely been described. In the present study, the postprandial patterns of net portal-drained viscera (PDV) and hepatic metabolism were assessed to evaluate purine and pyrimidine N in dairy cows. Blood was sampled simultaneously from four veins with eight hourly samples from four multi-catheterised Holstein cows. Quantification of twenty purines and pyrimidines was performed with HPLC-MS/MS, and net fluxes were estimated across the PDV, hepatic tissue and total splanchnic tissue (TSP). Concentration differences between veins of fifteen purine and pyrimidine nucleosides (NS), bases (BS) and degradation products (DP) were different from zero (P≤ 0·05), resulting in the net PDV releases of purine NS (0·33-1·3 mmol/h), purine BS (0·0023-0·018 mmol/h), purine DP (7·0-7·8 mmol/h), pyrimidine NS (0·30-2·8 mmol/h) and pyrimidine DP (0·047-0·77 mmol/h). The hepatic removal of purine and pyrimidine was almost equivalent to the net PDV release, resulting in no net TSP release. One exception was uric acid (7·9 mmol/h) from which a large net TSP release originated from the degradation of purine NS and BS. A small net TSP release of the pyrimidine DP ß-alanine and ß-aminoisobutyric acid (-0·032 to 0·37 mmol/h) demonstrated an outlet of N into the circulating N pool. No effect of time relative to feeding was observed (P>0·05). These data indicate that considerable amounts of N are lost in the dairy cow due to prominent intermediary degradation of purines, but that pyrimidine N is reusable to a larger extent.

Simultaneous quantification of purine and pyrimidine bases, nucleosides and their degradation products in bovine blood plasma by high performance liquid chromatography tandem mass spectrometry.

Improved nitrogen utilization in cattle is important in order to secure a sustainable cattle production. As purines and pyrimidines (PP) constitute an appreciable part of rumen nitrogen, an improved understanding of the absorption and intermediary metabolism of PP is essential. The present work describes the development and validation of a sensitive and specific method for simultaneous determination of 20 purines (adenine, guanine, guanosine, inosine, 2'-deoxyguanosine, 2'-deoxyinosine, xanthine, hypoxanthine), pyrimidines (cytosine, thymine, uracil, cytidine, uridine, thymidine, 2'-deoxyuridine), and their degradation products (uric acid, allantoin, ß-alanine, ß-ureidopropionic acid, ß-aminoisobutyric acid) in blood plasma of dairy cows. The high performance liquid chromatography-based technique coupled to electrospray ionization tandem mass spectrometry (LC-MS/MS) was combined with individual matrix-matched calibration standards and stable isotopically labelled reference compounds. The quantitative analysis was preceded by a novel pre-treatment procedure consisting of ethanol precipitation, filtration, evaporation and reconstitution. Parameters for separation and detection during the LC-MS/MS analysis were investigated. It was confirmed that using a log-calibration model rather than a linear calibration model resulted in lower CV% and a lack of fit test demonstrated a satisfying linear regression. The method covers concentration ranges for each metabolite according to that in actual samples, e.g. guanine: 0.10-5.0 µmol/L, and allantoin: 120-500 µmol/L. The CV% for the chosen quantification ranges were below 25%. The method has good repeatability (CV%≤25%) and intermediate precision (CV%≤25%) and excellent recoveries (91-107%). All metabolites demonstrated good long-term stability and good stability within-runs (CV%≤10%). Different degrees of absolute matrix effects were observed in plasma, urine and milk. The determination of relative matrix effects revealed that the method was suitable for almost all examined PP metabolites in plasma drawn from an artery and the portal hepatic, hepatic and gastrosplenic veins and, with a few exceptions, also for other species such as chicken, pig, mink, human and rat.