Regulation of aromatase P450 expression by puerarin in endometrial cell line RL95-2.
OBJECTIVE
To study the effects of puerarin on the aromatase P450 (P450(arom)) mRNA expression and the effects of low-dose puerarin on transcription factors of the P450(arom) gene (P II) 5'-flanking region.


METHODS
The effects of puerarin on the P450(arom) mRNA expression were determined by real-time polymerase chain reaction (RT-PCR). The 5'-flanking region was amplified by PCR using human genomic cDNA as a template. By means of the restriction sites and sequence confirmation, the PCR product was cloned into reporter vector. Series of sequential deletion reporter constructs were transiently transfected into RL95-2 cells which were treated with or without puerarin. Luciferase activity was measured by Dual-Luciferase Reporter Assay System and Luminoskan Ascent luminometer. Furthermore, by using web-based search program, the most possible cis-acting elements and transcription factors were evaluated.


RESULTS
The data demonstrated that low-dose puerarin treatment could decrease P450(arom) expression at mRNA level compared to dimethyl sulphoxide (DMSO) treatment (P<0.01), and puerarin (10(-7)mol/L) had a time-course effect on P450(arom) mRNA expression, which reached the bottom at 12h (P<0.01). Cells transfected with the -763/+8 bp constructs showed decrease in relative luciferase activity after puerarin (10(-7)mol/L) treatment compared to DMSO treatment (P<0.05), indicating an essential regulatory site between -763 bp and -543 bp responsible for the transcription suppression by puerarin. Furthermore, the most possible transcription factors, which turned out to be AP-1(c-jun/c-fos) at -410/-401 bp were also evaluated. The activity of exogenous AP-1 was reduced after 12 hours of puerarin treatment (P<0.05). The inhibition of c-jun mRNA also showed a time-course effect, which bottomed out at 12h in parallel with that of P450(arom) (P<0.01). The protein level of c-jun was also down-regulated by puerarin (10(-7)mol/L) treatment at 12h.


CONCLUSIONS
The suppression of P450(arom) expression and activity may be associated with the down-regulation of transcription factor AP-1/c-jun. This partially explains the mechanisms whereby puerarin treats endometriosis.
Department of Traditional Chinese Medicine, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
Yan-Wei
YW
Li
Zai-Long
ZL
Cai
Wei
W
Shen
Sheng-Sheng
SS
Yang
Chao-Qin
CQ
Yu
D001141:Aromatase / Q000235:genetics / Q000378:metabolism*; D047072:Aromatase Inhibitors / Q000494:pharmacology*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects; D004717:Endometrium / Q000166:cytology* / Q000201:enzymology*; D005260:Female; D006801:Humans; D007529:Isoflavones / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000187:drug effects
D047072:Aromatase Inhibitors; D007529:Isoflavones; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D001141:Aromatase; C033607:puerarin