Establishment of type II 5alpha-reductase over-expressing cell line as an inhibitor screening model.
Dihydrotestosterone (DHT) is the most potent male hormone that causes androgenetic alopecia. The type II 5alpha-reductase is an enzyme that catalyzes the conversion of testosterone (T) to DHT, therefore it can be expected that specific inhibitors for type II 5alpha-reductase may improve the pathophysiologic status of androgenetic alopecia. In this study, we attempted to establish the reliable and convenient screening model for type II 5alpha-reductase inhibitors. After transfection of human cDNA for type II 5alpha-reductase into HEK293 cells, the type II 5alpha-reductase over-expressing stable cells were selected by G418 treatment. RT-PCR and Western blot analyses confirmed that type II 5alpha-reductase gene was expressed in the stable cells. In in vitro enzymatic assay, 10 microg of stable cell extract completely converted 1 microCi (approximately 0.015 nmol) of T into DHT. The type II 5alpha-reductase activity was inhibited by finasteride in a dose-dependent manner, confirming the reliability of screening system. In cell culture condition, 2 x 10(5) of stable cells completely converted all the input T (approximately 0.03 nmol) into DHT by 4h incubation, demonstrating that the stable cell line can be used as a cell-based assay system. Using this system, we selected the extracts of Curcumae longae rhizoma and Mori ramulus as the potential inhibitors for type II 5alpha-reductase. These results demonstrate that the type II 5alpha-reductase over-expressing stable cell line is a convenient and reliable model for screening and evaluation of inhibitors.
Jang
Sunhyae
S
Department of Dermatology and Research Institute for Medical Sciences, School of Medicine, Chungnam National University, Daejeon, Republic of Korea.
Lee
Young
Y
Hwang
Seong-Lok
SL
Lee
Min-Ho
MH
Park
Su Jin
SJ
Lee
In Ho
IH
Kang
Sangjin
S
Roh
Seok-Seon
SS
Seo
Young-Joon
YJ
Park
Jang-Kyu
JK
Lee
Jeung-Hoon
JH
Kim
Chang Deok
CD
D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D042944:Cholestenone 5 alpha-Reductase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D008962:Models, Theoretical; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D004791:Enzyme Inhibitors; D042944:Cholestenone 5 alpha-Reductase