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W. Samuel, R. K. Kutty, T. Duncan, T. M. Redmond; Inhibition of N-(4-Hydroxyphenyl)retinamide-Induced Apoptosis in Human Retinal Pigment Epithelial Cells by Sterculic Acid, a Stearoyl Coenzyme A Desaturase Inhibitor. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4815.
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Apoptosis in retinal pigment epithelium (RPE) due to oxidative stress could hasten the onset of age-related macular degeneration. Recently, we showed that N-(4-hydroxyphenyl)retinamide (4HPR), a retinoic acid (RA) derivative, induces apoptosis in human RPE cells via reactive oxygen species (ROS). Earlier, we showed in these cells that RA regulates the expression of stearoyl coenzyme A desaturase (SCD), an enzyme that regulates cell growth, differentiation and apoptosis by controlling the ratio of saturated to unsaturated fatty acids. Therefore, in the present study, we investigated the effect of sterculic acid, a known inhibitor of SCD, on 4HPR-induced apoptosis in human RPE cells.
Human RPE cells (ARPE-19) in culture were treated with 4HPR in the presence or absence of sterculic acid for various time intervals. The progression of apoptosis was followed by a sandwich-enzyme immunoassay using mouse monoclonal antibodies directed against DNA and histones. Generation of ROS was measured fluorimetrically using carboxy-H2DCFDA. The expression of GADD153 and heme oxygenase-1 (HO-1) was measured using real-time RT-PCR.
4HPR induced apoptosis in ARPE-19 cells in a dose- and time-dependent manner as indicated by the generation of mono-and oligonucelosomes. Sterculic acid, the SCD inhibitor, blocked the 4HPR-induced apoptosis by ~90%. The ROS generation induced by 4HPR was also effectively suppressed by sterculic acid. The 4HPR treatment increased GADD153 and HO-1 expression by ~11-and ~95-fold, respectively. This increase was completely blocked by the SCD inhibitor. A four-fold increase in the activity of caspase-2 and caspase-3 observed with 4HPR-treatment was also abolished by sterculic acid.
Our results show that sterculic acid effectively blocked 4HPR induced apoptosis in human RPE cells. The mode of action of this SCD inhibitor appears to be at the level of ROS generation. Thus, SCD may play an important role in regulating the response of RPE to oxidative stress.
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