N-(4-hydroxyphenyl)retinamide activation of a distinct pathway signaling apoptosis.

The clinical and basic scientific understanding of the role of retinoids in cancer therapy and prevention has seen advances. Retinoids are synthetic and natural analogues of vitamin A [reviewed in (1)]. These compounds are ligands for the retinoid receptors, members of the steroid receptor superfamily (2,3). The biologic effects of retinoids are initiated through liganddependent activation of nuclear retinoid receptors. This interaction leads, in turn, to activation or repression of ‘‘target genes,’’ which results in growth and differentiation effects. The finding that the retinoid N-(4-hydroxyphenyl)retinamide (4HPR) induces apoptosis in cells resistant to all-trans-retinoic acid, a high-affinity ligand for the retinoic acid receptors (RARs) (2,3), has suggested that 4HPR signals through a mechanism independent of the retinoid receptors (4–7). In this issue of the Journal, an article by Oridate et al. (8) links the generation of reactive oxygen species to 4HPR-induced apoptosis in cervical cancer cells. Confirmation that this pathway is active in other cell contexts would provide important evidence for a novel retinoid mechanism signaling apoptosis in human tumor cells. Retinoids have reported activity in cancer therapy and prevention. They have been used in the treatment of the premalignant lesion oral leukoplakia (9) and in the prevention of second cancers of the head and neck (10), lung (11), and liver (12). As a single agent, all-trans-retinoic acid induces transient complete remissions in acute promyelocytic leukemia (13–15). When combined with interferon alfa-2a treatment, 13-cis-retinoic acid induces complete and partial remissions in squamous cell carcinoma of the skin and cervix (16,17). This regimen is also reported to be active in the treatment of disseminated renal cancer (18). Retinoid biologic effects are signaled through two families of structurally related receptors, the RARs and the retinoid X receptors (RXRs) (1,2). The RARs have three subtypes (RAR a, RAR b, and RAR g), with multiple isoforms. The RXRs also have three family members (RXR a, RXR b, and RXR g). Orphan nuclear receptors exist for which physiologic ligands are not yet identified. All-trans-retinoic acid activates transcription of the RAR pathway but not the RXR pathway. Retinoid receptor ligands have been engineered to activate or inhibit specific RAR or RXR family members (19). Other retinoid ligands antagonize AP-1 activity (20) to signal biologic effects. While 4HPR is reported to activate transcription by specific retinoid receptors, including RAR g (21), the signaling of apoptosis by 4HPR in retinoid-resistant tumor cells (4) raises the prospect that 4HPR mediates its effects through a retinoid receptorindependent mechanism. The finding that 4HPR signals apoptosis through generation of reactive oxygen species (8) is consistent with this interpretation. Since 4HPR activity is seen at concentrations expected for a receptor-dependent pathway, perhaps some receptor-mediated signals contribute to the observed biologic effects. A convergence exists between clinical and basic scientific findings in the retinoid field. This convergence is illustrated in the link between retinoid-induced RAR b expression and clinical responses in oral leukoplakia (22) and in the association between PML/RAR a expression and all-trans-retinoic acidmediated remissions in acute promyelocytic leukemia (24). These examples underscore the importance of understanding which retinoid pathways are engaged to signal biologic and clinical effects. The study by Oridate et al. (8) extends previous work on 4HPR by highlighting a distinct retinoid mechanism signaling apoptosis independent of nuclear receptors. This mechanism is the accumulation of reactive oxygen species induced by 4HPR treatment. Several novel findings are reported by Oridate et al. 4HPR signals apoptosis in malignant but not in benign cervical epithelial cells. The specificity of the 4HPR pro-oxidant effect is supported by the finding that the major metabolite of 4HPR does not signal apoptosis or generate reactive oxygen species. 4HPR does not appear to alter expression of the Bcl-2 or Bax proteins. These species and p53 are linked to the regulation of apoptosis in vivo (25). Treatment with oxygen radical scavengers reduces reactive oxygen species and inhibits 4HPR-induced apoptosis. A threshold effect is observed, indicating a critical level of reactive oxygen species is needed to generate apoptosis. Notably, while 4HPR is reported to activate transcription by RAR g (21), treatment with an RAR g antagonist did not prevent 4HPR signaling. This finding is consistent with the proposed retinoid receptorindependent mechanism. The tight link between reactive oxygen species and 4HPR