Biochemical and cellular consequences of lumiflavin-induced riboflavin depletion in human epithelial cells

Riboflavin is an essential component of the human diet, with an established role for its derivative cofactors, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), in oxidative metabolism. It has previously been demonstrated that luminal but not systemic riboflavin depletion leads to a dysregulation of normal gastrointestinal development. It is hypothesised that riboflavin depletion of intestinal cells in culture interrupts cell signalling pathways, with associated adverse functional effects. The overall aim was to develop and characterise an intestinal cell model of riboflavin depletion using the structural analogue of riboflavin, lumiflavin, which inhibits riboflavin uptake, and to assess the role of riboflavin on cell signalling. A model of riboflavin depletion was established in three intestinal cell lines, (Caco-2, HCT116 and HT29) through treatment with lumiflavin. Intracellular flavin concentrations and glutathione reductase activation coefficient (GRAC) were measured as markers of riboflavin status. Intracellular ATP concentration, the generation of reactive oxygen species (ROS), clonogenicity and apoptotic DNA fragmentation were determined. Effects of riboflavin depletion on cell signalling were examined in only Caco-2 cells. Changes in the phosphorylation of amino acid residues were determined as an indicator of effects on cell signalling, using western blot and high content analysis (HCA). Isobaric tagging for relative and absolute quantitation (iTRAQ) was used for phosphoprotein profiling, followed by validation of the findings by western blot. HCA and flow cytometry were used to determine changes in the cell cycle. Cell growth was inhibited by lumiflavin in all three cell lines, in a concentration-dependent manner. Intracellular flavin status was significantly decreased in all cell lines by 48 hours. Riboflavin depletion by lumiflavin led to a significant reduction in intracellular ATP concentration and an enhanced generation of ROS in all cell lines. A cell-specific irreversible loss of proliferative ability was observed. The effect of riboflavin depletion on oxidative stress and disruption of energy generation may have contributed to the observed effects on cell proliferation. There was no effect of lumiflavin on apoptotic DNA fragmentation in any cell line. Lumiflavin resulted in changes in the global phosphorylation of tyrosine by 3-6 hours, before biochemical riboflavin depletion was evident. Significant changes in mRNA processing and apoptosis pathways were found. Lumiflavin seemed to suppress cell signalling. Lumiflavin also caused arrest of cells in S phase with a subsequent G2/M phase block. In conclusion, riboflavin depletion of intestinal cells by lumiflavin had biochemical and functional consequences. Alterations in cell signalling and arrest in S phase with induction of apoptosis in response to lumiflavin may be causative of adverse consequences on intestinal cell proliferation