HepG2 cells develop signs of riboflavin deficiency within 4 days of culture in riboflavin-deficient medium

Abstract

Flavin mononucleotide and flavin adenine dinucleotide are essential coenzymes in redox reactions. For example, flavin adenine dinucleotide is a coenzyme for both glutathione reductase and enzymes that mediate the oxidative folding of secretory proteins. Here we investigated short-term effects of moderately riboflavin-deficient culture medium on flavin-related responses in HepG2 hepatocarcinoma cells. Cells were cultured in riboflavin-deficient (3.1 nmol/L) medium for up to six days; controls were cultured in riboflavin-sufficient (532 nmol/L) medium. The activity of glutathione reductase decreased by 98% within four days of riboflavin-deficient culture. Transport rates of riboflavin increased in response to riboflavin depletion, whereas expression of enzymes mediating flavocoenzyme synthesis (flavokinase and flavin adenine dinucleotide synthetase) decreased in response to depletion. The oxidative folding and synthesis of plasminogen and apolipoprotein B-100, respectively, was impaired within four days of culture in riboflavin-deficient medium; this is consistent with impaired processing of secretory proteins in riboflavin-deficient cells. Riboflavin depletion was associated with increased DNA-binding activities of transcription factors with affinity for endoplasmic reticulum stress elements and NF-κB consensus elements, suggesting cell stress. Moreover, the abundance of the stress-induced protein GADD153 was greater in riboflavin-deficient cells compared with controls. Riboflavin deficiency was associated with decreased rates of cell proliferation caused by arrest in G1 phase of the cell cycle. These studies are consistent with the hypothesis that HepG2 cells have a great demand for riboflavin, and that cell stress develops rapidly if riboflavin supply is marginally low.

Riboflavin Depletion Impairs Cell Proliferation in Adult Human Duodenum: Identification of Potential Effectors

“…The cell growth rate decreases, and the cells start to slow their passage through G2 and M. Eventually, when the cells are severely depleted in riboflavin, growth slows and this is associated with an increase in cells at G2/M. This is in contrast with HepG2 cells, which also showed a reduced growth rate on riboflavin depletion, but arrested in G1 [14,36].…”

“…At riboflavin concentrations in the medium corresponding to low plasma concentrations in humans, intracellular levels of FMN and FAD cannot be maintained. It has been shown in other cell lines that cellular flavin depletion causes decrease in activity of flavin-dependent enzymes such as the flavoprotein Ero1p, which is essential for oxidative folding of proteins in the endoplasmic reticulum (ER) in yeast [11], and glutathione reductase, which generates the antioxidant reduced glutathione [13,14]. Thus, a fall in intracellular FMN and FAD concentration can lead to oxidative stress, which is associated with an increased rate of apoptosis and arrest of the cell cycle [13].…”

“…In Jurkat lymphoid cells, riboflavin depletion has rather little effect on cell viability [11]. In contrast, in HepG2 human liver cells, riboflavin deficiency leads to an unfolded protein stress response [12] and arrest in G1 [13,14], as shown by flow cytometry. In rat fibroblasts and mouse 3T3 cells, addition of the flavoprotein inhibitor diphenyleneiodonium led to arrest in G2 [15].…”

Low activity of LSD1 elicits a pro-inflammatory gene expression profile in riboflavin-deficient human T Lymphoma Jurkat cells

“…Cells were cultured in riboflavin-sufficient medium for 7 days before transfer into the riboflavin-defined media and continued culture for 7 days before analysis. This protocol was chosen based on previous and preliminary studies, suggesting abnormally slow cell growth after 10 days of culture in riboflavin-deficient medium Werner et al 2005). The production of pro-inflammatory cytokines was induced by treatment with 100 lg/L phorbol 12-myristate 13-acetate (PMA) for 8 h before analysis (Khalaf et al 2010).…”

Involvement of riboflavin kinase expression in cellular sensitivity against cisplatin

“…3, down-regulation of RFK reduced cell growth and induced apoptosis. On the other hand, riboflavin deficiency induced G1 cell cycle arrest in HepG2 cells (26). Recently, it has been reported that RFK interacts with TNFR1 (tumour necrosis factor receptor 1) and is involved in TNF (tumour necrosis factor)-dependent signal transduction (27).…”