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Grace B. Woo, Laura S. Woo, Claudio Ramirez, Khoa Pham, Sarah Hashmi, Marilyn Chwa, Baruch D. Kuppermann, Maria C. Kenney; Hydroquinone Induces Autophagy in Human Retinal Pigment Epithelial Cells in vitro. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4276.
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© ARVO (1962-2015); The Authors (2016-present)
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.
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.
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.
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.
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