Abstract
Purpose: :
We explored the effects of hydroquinone (HQ), a toxic component found in cigarette tar, on human retinal pigment epithelial cells (ARPE-19) in order to understand the mechanism(s) of cell death.
Methods: :
ARPE-19 cells were grown in vitro and treated for 24 hours with four different concentrations of HQ (50μM, 100μM, 200μM, 500μM). Caspase 3/7 activity was detected using the Carboxyfluorescein FLICA Apoptosis Detection kit. Proteins were extracted and subjected to Western blot analyses probed with rabbit anti-microtubule-associated protein light chain 3 (LC3-II), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), mouse anti-HSP60 (Heat Shock Protein 60), rabbit anti-HSP70 (Heat Shock Protein 70), and mouse anti-NMDAR1 (N-methyl-D-aspartate receptor 1). The resulting bands were visualized via colorimetric detection, quantified using ImageJ, and subjected to statistical analysis using GraphPad Prism software.
Results: :
After treatment with HQ, there was no change in caspase 3/7 activities at any of the concentrations tested. Both HSP60 and NMDAR1 levels were significantly decreased at 200μM HQ (5860±601 vs 11394±1670, p=0.0110; 1814±218 vs 7717±855, p=0.0478, respectively) while the HSP70 levels increased (24209±2298 vs 13605±2582, p=0.049) at 100μM HQ compared to the dimethyl sulfoxide (DMSO) treated controls. The LC3-II band (14kDa) was present in the cultures treated with 100μM and 200μM HQ.
Conclusions: :
Our findings suggest that the mechanism of cell death for ARPE-19 cells exposed to HQ does not involve apoptosis but occurs via autophagy, as confirmed by the presence of LC3-II, and other cell-signaling molecules. The HSP70, which has been implicated as an anti-apoptotic and pro-autophagic signaling molecule, was elevated and HSP60, which has been implicated as pro-apoptotic, was decreased following HQ exposure. The reduced NMDAR1 levels suggest that the disruption of glutamate homeostasis may also play a role in the HQ-induced cell death.
Keywords: apoptosis/cell death • age-related macular degeneration • stress response