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. CONCLUSION: 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.