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.
Hursting
Stephen D
SD
Laboratory of Biosystems and Cancer, National Cancer Institute at Frederick, Frederick, Maryland, USA.
Shen
Jian-cheng
JC
Sun
Xiao-Ya
XY
Wang
Thomas T Y
TT
Phang
James M
JM
Perkins
Susan N
SN
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D015152:Blotting, Northern; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism*; D021983:Cyclophilins / Q000096:biosynthesis / Q000235:genetics*; D017313:Fenretinide / Q000494:pharmacology*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008297:Male; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics; D016133:Polymerase Chain Reaction; D011471:Prostatic Neoplasms / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D012334:RNA, Neoplasm / Q000096:biosynthesis; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000187:drug effects
D000970:Antineoplastic Agents; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D017382:Reactive Oxygen Species; D017313:Fenretinide; D021983:Cyclophilins