Figure 4
Flavonoids and calpeptin decrease the inhibitory effect of Cdk5 on PXR-mediated CYP3A4 promoter activity. (A) HepG2 was transiently co-transfected with FLAG-PXR, CYP3A4-luc, CMV-renilla luciferase (as a transfection control), and either Cdk5 (+ Cdk5) or pcDNA3 vector (-Cdk5) plasmids at a ratio of 1:15:1:3, respectively. A total of 20 μg of combined plasmids was used to transfect 5 × 106 cells seeded in a T75 flask 16 h before transfection. Transfected cells were seeded into white 96-well plates (at 10,000 cells/well) 24 h later, and treated with DMSO or compounds as indicated for 24 h before Dual-glo luciferase assay. CYP3A4 promoter activity was normalized by using activity of the CMV-renilla, and expressed as fold induction over control (DMSO; without Cdk5 transfection). The values represent the average of 8 independent experiments, with the standard deviation denoted as bars. The significance of the difference between datasets was determined by using the Student's t test. (B) Using fold induction over control as described in (A), the Cdk5-mediated inhibition of CYP3A4 promoter activity was expressed a percentage of inhibition by Cdk5 [% inhibition = 100% × (signal without Cdk5 - signal with Cdk5)/signal without Cdk5)].