Modulation of cyclophilin gene expression by N-4-(hydroxyphenyl)retinamide: association with reactive oxygen species generation and apoptosis.

To explore the mechanisms underlying the pro-apoptotic effects of the synthetic retinoid N-4-(hydroxyphenyl)retinamide (4-HPR) on LNCaP human prostate cancer cells, we used the differential display-polymerase chain reaction (DD-PCR) technique to identify 4-HPR-responsive genes. RNA extracted from LNCaP cells that had been treated for 24 h with 4-HPR at a dose (2.5 microM) optimal for apoptosis induction was used for DD-PCR analysis using random primers. A differentially expressed 115 bp fragment was cloned and sequenced and then identified in GenBank as having a high degree of homology with several members of the cyclophilin gene family. Northern blot analyses using specific probes for cyclophilin A, cyclophilin D, and the cloned 115-bp fragment were performed on RNA extracted from LNCaP cells and MCF-7 human breast cancer cells treated with 4-HPR, N-acetylcysteine (NAC, an anti-oxidant), 4-HPR plus NAC, cyclosporin A, R-1881 (a synthetic androgen), dehydroepiandrosterone, all-trans retinoic acid, or prednisone. 4-HPR downregulated the transcript detected by the 115-bp fragment. Expression patterns detected by the 115-bp fragment and cyclophilin D probes were identical in response to each treatment; none of these treatments affected cyclophilin A expression. Furthermore, expression of mRNA transcripts detected by the 115-bp fragment and cyclophilin D probes correlated with the generation of reactive oxygen species (ROS), as detected by measurement of 2,7-dichlorofluorescein oxidation. Therefore, members of the cyclophilin gene family, such as cyclophilin D (a component of the mitochondrial permeability transition pore previously linked with oxidative stress and apoptosis), may play a role in the ROS-mediated apoptotic effects of 4-HPR.