Increasing Intracellular Levels of Iron with Ferric Ammonium Citrate Leads to Reduced P-glycoprotein Expression in Human Immortalised Brain Microvascular Endothelial Cells

Abstract

Purpose P-glycoprotein (P-gp) at the blood-brain barrier (BBB) precludes the brain penetration of many xenobiotics and mediates brain-to-blood clearance of β-amyloid, which accumulates in the Alzheimer’s disease (AD) brain. Zinc and copper are reported to modulate BBB expression and function of P-gp; however, the impact of exogenous iron, which accumulates in AD, on P-gp dynamics remains unknown. Methods P-gp protein and MDR1 transcript levels were assessed in immortalised human cerebral microvascular endothelial (hCMEC/D3) cells treated with ferric ammonium citrate (FAC; 250 μM, 72 h), by Western blotting and RT-qPCR, respectively. P-gp function was assessed using rhodamine-123 and [³H]-digoxin accumulation. Intracellular reactive oxygen species (ROS) levels were determined using 2′,7′-dichlorofluorescin diacetate and intracellular iron levels quantified using a ferrozine assay. Results FAC treatment significantly reduced P-gp protein (36%) and MDR1 mRNA (16%) levels, with no significant change in rhodamine-123 or [³H]-digoxin accumulation. While P-gp/MDR1 downregulation was associated with elevated ROS and intracellular iron, MDR1 downregulation was not attenuated with the antioxidant N-acetylcysteine nor the iron chelators desferrioxamine and deferiprone, suggesting the involvement of a ROS-independent mechanism or incomplete iron chelation. Conclusions These studies demonstrate that iron negatively regulates P-gp expression at the BBB, potentially impacting CNS drug delivery and brain β-amyloid clearance.