Glutathione depletion regulates both extrinsic and intrinsic apoptotic signaling cascades independent from multidrug resistance protein 1.
ABSTRACT Glutathione (GSH) depletion is an important hallmark of apoptosis. We previously demonstrated that GSH depletion, by its efflux, regulates apoptosis by modulation of executioner caspase activity. However, both the molecular identity of the GSH transporter(s) involved and the signaling cascades regulating GSH loss remain obscure. We sought to determine the role of multidrug resistance protein 1 (MRP1) in GSH depletion and its regulatory role on extrinsic and intrinsic pathways of apoptosis. In human lymphoma cells, GSH depletion was stimulated rather than inhibited by pharmacological blockage of MRP1 with MK571. GSH loss was dependent on initiator caspases 8 and 9 activity. Genetic knock-down (>60 %) of MRP1 by stable transfection with short hairpin small interfering RNA significantly reduced MRP1 protein levels, which correlated directly with the loss of MRP1-mediated anion transport. However, GSH depletion and apoptosis induced by both extrinsic and intrinsic pathways were not affected by MRP1 knock-down. Interestingly, stimulation of GSH loss by MK571 also enhanced the initiator phase of apoptosis by stimulating initiator caspase 8 and 9 activity and pro-apoptotic BCL-2 interacting domain cleavage. Our results clearly show that caspase-dependent GSH loss and apoptosis are not mediated by MRP1 proteins and that GSH depletion stimulates the initiation phase of apoptosis in lymphoid cells.
SourceAvailable from: Yahya Sefidbakht[Show abstract] [Hide abstract]
ABSTRACT: The effects of mobile phone frequency electromagnetic field (RF-EMF, 940 MHz) on a stable cell line (HEK293T) harbouring firefly luciferase gene were evaluated. A waveguide exposure system with 1W input power provided the mean specific absorption rate of [approximate] 0.09 W/kg in 35 mm Petri dishes. The effects of exposure duration (15, 30, 45, 60 and 90 min) were investigated on luciferase activity and oxidative response elements. Endogenous luciferase activity reduced after 30 and 45 min continuous exposures. While, after 60 min, the exposed cell lysate showed higher luciferase activity compared with the non-exposed control. Reactive oxygen species (ROS) generation was highest in the 30 min exposed cells as studied by DCFH-DA fluorescence. The observed boost in ROS was then followed by a sharp rise in catalase (CAT) and superoxide dismutase (SOD) activity and elevation of glutathione (GSH) during the 45 min exposure. Decrease in lipid peroxidation (malondialdehyde, MDA) was meaningful for the 45 andPhotochemical and Photobiological Sciences 01/2014; DOI:10.1039/C3PP50451D · 2.94 Impact Factor