N-(4-hydroxyphenyl)retinamide induces apoptosis in human retinal pigment epithelial cells: retinoic acid receptors regulate apoptosis, reactive oxygen species generation, and the expression of heme oxygenase-1 and Gadd153.

N-(4-hydroxyphenyl)retinamide (4HPR, fenretinide), a retinoic acid (RA) derivative and a potential cancer preventive agent, is known to exert its chemotherapeutic effects in cancer cells through induction of apoptosis. Earlier work from our laboratory has shown that relatively low concentrations of 4HPR induce neuronal differentiation of cultured human retinal pigment epithelial (ARPE-19) cells (Chen et al., 2003, J Neurochem 84:972-981). However, at higher concentrations of 4HPR, these cells showed morphological changes including cell shrinkage and cell death. Here we demonstrate that ARPE-19 cells treated with 4HPR exhibit a dose- and time-dependent induction of apoptosis as evidenced by morphological changes, mono- and oligonucleosome generation, and increased activity of caspases 2 and 3. The 4HPR-induced apoptosis as well as the activation of caspases 2 and 3 were blocked by both retinoic acid receptors (RAR) pan-antagonists, AGN193109 and AGN194310, and by an RARalpha-specific antagonist AGN194301. 4HPR treatment also increased reactive oxygen species (ROS) generation in ARPE-19 cells in a time-dependent manner as determined from the oxidation of 2',7'-dichlorofluorescin. In addition, the increase in the expression of heme oxygenase-1 (HO-1), a stress response protein, and the growth arrest and DNA damage-inducible transcription factor 153 (Gadd153) in response to the ROS generation were also blocked by these receptor antagonists. Pyrrolidine dithiocarbamate (PDTC), a free-radical scavenger, inhibited 4HPR-induced ROS generation, the expression of its downstream mediator, Gadd153, and apoptosis in the pretreated cells. Therefore, our results, clearly demonstrate that 4HPR induces apoptosis in ARPE-19 cells and that RARs mediate this process by regulating ROS generation as well as the expression of Gadd153 and HO-1.