Inhibition of N–(4–Hydroxyphenyl)Retinamide–Induced Apoptosis of Human Retinal Pigment Epithelial Cells by 9–Thiastearate, a Stearoyl Coenzyme a Desaturase Inhibitor

Abstract

Abstract: : Purpose: Retinoic acid, a natural derivative of vitamin A, and its synthetic analogues have a profound effect on many cellular functions including cell growth, differentiation, and apoptosis. Among them, N–(4–hydroxyphenyl)retinamide (4HPR, fenretinide), an amide analog of retinoic acid, exerts its chemotherapeutic effects on cancer cells through induction of apoptosis. Recently, we have shown that 4HPR induces apoptosis in human RPE (ARPE–19) cells. Earlier, we have found that retinoic acid regulates the expression of stearoyl–CoA desaturase (SCD) in these cells. SCD, a microsomal enzyme catalyzing the initial desaturation of long chain fatty acids into monounsaturated fatty acids, is thought to play an important role in cell growth, differentiation and apoptosis by its ability to control the ratio of saturated to unsaturated fatty acids in cells. In addition certain fatty acid analogues, such as thia–fatty acids, are known to exert antineoplastic activity by inhibiting SCD. Therefore, in the present study, we investigated the effect of 9–thiastearate, an inhibitor of SCD, on 4HPR–induced apoptosis of human RPE cells. Methods: Human RPE cells (ARPE–19) in culture were treated with 4HPR in the presence or absence of the SCD inhibitor, 9–thiastearate, for various time intervals. Cell lysates were used to measure the progression of apoptosis by a sandwich–enzyme immunoassay using anti–histone antibody directed against mono–and oligonucleosomes. Activities of caspase–2 and caspase–3, members of cysteine proteases involved in apoptosis, were measured using a fluorimetric method. Results: 4HPR induced apoptosis in ARPE–19 cells in a dose– and time–dependent manner as indicated by the generation of mono– and oligonucleosomes. 9–thiastearate, a specific inhibitor of SCD, effectively blocked the apoptosis. 4HPR–induced apoptosis was accompanied by the marked increase in the activities of caspase–2 and caspase–3. Further, the 4HPR–induced activation of caspases was effectively inhibited by 9–thiastearate. Conclusions: We have shown for the first time that 4HPR–induced apoptosis of ARPE–19 cells is blocked by 9–thiastearate, an inhibitor of SCD, indicating the involvement of this enzyme in the process.

Keywords: apoptosis/cell death • retinal pigment epithelium • cell death/apoptosis