Antiproliferative Effect of Ascorbic Acid Is Associated with the Inhibition of Genes Necessary to Cell Cycle Progression

BACKGROUND: Ascorbic acid (AA), or Vitamin C, is most well known as a nutritional supplement with antioxidant properties. Recently, we demonstrated that high concentrations of AA act on PMP22 gene expression and partially correct the Charcot-Marie-Tooth disease phenotype in a mouse model. This is due to the capacity of AA, but not other antioxidants, to down-modulate cAMP intracellular concentration by a competitive inhibition of the adenylate cyclase enzymatic activity. Because of the critical role of cAMP in intracellular signalling, we decided to explore the possibility that ascorbic acid could modulate the expression of other genes. METHODS AND FINDINGS: Using human pangenomic microarrays, we found that AA inhibited the expression of two categories of genes necessary for cell cycle progression, tRNA synthetases and translation initiation factor subunits. In in vitro assays, we demonstrated that AA induced the S-phase arrest of proliferative normal and tumor cells. Highest concentrations of AA leaded to necrotic cell death. However, quiescent cells were not susceptible to AA toxicity, suggesting the blockage of protein synthesis was mainly detrimental in metabolically-active cells. Using animal models, we found that high concentrations of AA inhibited tumor progression in nude mice grafted with HT29 cells (derived from human colon carcinoma). Consistently, expression of tRNA synthetases and ieF2 appeared to be specifically decreased in tumors upon AA treatment. CONCLUSIONS: AA has an antiproliferative activity, at elevated concentration that could be obtained using IV injection. This activity has been observed in vitro as well in vivo and likely results from the inhibition of expression of genes involved in protein synthesis. Implications for a clinical use in anticancer therapies will be discussed

Lipopolysaccharides Impair Insulin Gene Expression in Isolated Islets of Langerhans via Toll-Like Receptor-4 and NF-κB Signalling

BACKGROUND:Type 2 diabetes is characterized by pancreatic β-cell dysfunction and is associated with low-grade inflammation. Recent observations suggest that the signalling cascade activated by lipopolysaccharides (LPS) binding to Toll-Like Receptor 4 (TLR4) exerts deleterious effects on pancreatic β-cell function; however, the molecular mechanisms of these effects are incompletely understood. In this study, we tested the hypothesis that LPS alters insulin gene expression via TLR4 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in islets. METHODOLOGY/PRINCIPAL FINDINGS:A 24-h exposure of isolated human, rat and mouse islets of Langerhans to LPS dose-dependently reduced insulin gene expression. This was associated in mouse and rat islets with decreased mRNA expression of pancreas-duodenum homebox-1 (PDX-1) and mammalian homologue of avian MafA/l-Maf (MafA). Accordingly, LPS exposure also decreased glucose-induced insulin secretion. LPS repression of insulin, PDX-1 and MafA expression, as well as its inhibition of insulin secretion, were not observed in islets from TLR4-deficient mice. LPS inhibition of β-cell gene expression in rat islets was prevented by inhibition of the NF-κB pathway, but not the p38 mitogen-activated protein kinase (p38 MAPK) pathway. CONCLUSIONS/SIGNIFICANCE:Our findings demonstrate that LPS inhibit β-cell gene expression in a TLR4-dependent manner and via NF-κB signaling in pancreatic islets, suggesting a novel mechanism by which the gut microbiota might affect pancreatic β-cell function

Inhibition of NF-κB, but not p38 MAPK, restores insulin, PDX-1, and MafA gene expression in islets exposed to LPS.

<p>(A) Insulin pre-mRNA, (B) PDX-1 mRNA and (C) MafA mRNA expression in isolated rat islets exposed for 24 h to 16.7 mM (16.7 G) glucose in the presence or absence of 10 ng/mL LPS or 0.5 ng/mL IL-1β with or without SB202190 (10 µM) and IKK-2 Inh IV (10 µM). Data are mean ± S.E.M. of 3–4 independent experiments; *p<0.05.</p

PDX-1 cellular localization is not altered in response to LPS.

<p>HIT-T15 cells were transfected with a construct encoding a PDX-1-GFP fusion protein. PDX-1 localization (green) (A–F) was visualized by GFP fluorescence using a laser-scanning confocal microscope in cells cultured in 0.1 and 5 mM glucose with or without 0.5 mM palmitate or increasing doses of LPS. 4′,6-diamidino-2-phenylindole (DAPI) (blue) was used for nuclear staining (G–L). Images are representative of 3 replicate experiments.</p

Exposure to LPS dose-dependently represses insulin pre-mRNA expression in isolated rat and human islets.

<p>Insulin pre-mRNA levels in response to increasing doses of LPS in isolated rat (A) and human (B) islets. Pre-mRNA levels were measured by RT-PCR and normalized to β-actin mRNA levels. Data are mean ± S.E.M. of 2–6 independent experiments; *p<0.05 vs 0 pg/mL.</p

Potential mechanism by which LPS repress insulin gene expression in isolated islets.

<p>Exposure to LPS activates the NF-κB pathway in isolated islets and inhibits the expression of insulin, PDX-1 and MafA. The decrease in insulin expression might indirectly results from LPS inhibition of PDX-1 and MafA. NF-κB could also inhibit insulin gene expression by interacting with other proteins such as C/EBPβ and/or CREB, as observed in other cell types.</p