Abstract
Abstract: :
Purpose:The retinal pigment epithelium (RPE), a monolayer of highly differentiated epithelial cells located between the photoreceptors and choriocapillaris, is exposed to variety of stress, including exposure to light, inflammatory mediators, and reactive oxygen species. The integrity of the RPE layer, especially that of the macular region, is critical for the retention of normal visual function during the aging process. Apoptotic RPE cell death resulting from increased oxidative stress could hasten the onset of age–related macular degeneration (AMRD). Fenretinide, N–(4–hydoxyphenyl)retinamide, a retinoic acid (RA) derivative and a potential cancer preventive agent, exerts its chemotherapeutic effect in cancer cells through induction of apoptosis. The present study is aimed at understanding the role of reactive oxygen species in fenretinide–induced cell death in RPE cells. Methods:Human RPE cells (ARPE–19) in culture were treated with 1–20 µM fenretinide in the presence or absence of retinoid receptor antagonists for various time intervals. Cell lysates were used to measure the progression of apoptosis by a sandwich–enzyme immunoassay using anti–histone antibodies directed against mono–and oligonucleosomes. The intracellular generation of ROS was measured using carboxydichlorodihydrofluorescein diacetate, a nonfluorescent dye, which is oxidized inside the cell to fluorescent carboxydichlorofluorescein by reactive oxygen species. The rate of oxidation of the dye in the treated cells was measured using a fluorescence plate reader. Results:Fenretinide induced apoptosis in ARPE–19 cells in a dose– and time–dependent manner as indicated by the generation of mono– and oligonucleosomes. ROS generation was also increased in a dose– and time –dependent manner in the fenretinide treated ARPE–19 cells. Pyrrolidinedithiocarbamate (PDTC), a free–radical scavenger, inhibited the fenretinide induced apoptosis. AGN194301, a RARα receptor specific antagonist, effectively blocked the generation of ROS as well as apoptosis in the fenretinide treated cells. Conclusions:These results suggest that fenretinide induced apoptosis in RPE cells is mediated by ROS. Interestingly, both apoptosis and ROS generation are linked to the RARα–dependent signaling pathway. In the adult retina, the RPE is the major site of RA production from all–trans retinol generated during the visual process. Thus, the potential exists for the formation of toxic levels of ROS via the retinoid signaling pathway.
Keywords: apoptosis/cell death • oxidation/oxidative or free radical damage • retinoids/retinoid binding proteins