Figure 2

Berberine specifically inhibits AP-1 DNA binding activity in HPV-16 positive cervical cancer cells. SiHa cells treated with indicated concentrations of berberine for 24 h (A), or treated with 250 μg/ml berberine for indicated durations (B) were assayed for AP-1 DNA binding activity by EMSA and fold change in DNA binding was calculated by densitometric evaluation of shifted band. C, Binding of AP-1 to its consensus oligo probe is specific. Untreated SiHa nuclear proteins and labeled AP-1 probe were incubated in the presence of 100 fold molar excess of unlabeled homologous AP-1 probe or heterologous Oct-1 probe and assayed for AP-1 specific binding by EMSA. D, Berberine does not alter basal activity of other ubiquitous transcription factor Sp1. Nuclear proteins (10 μg) of berberine- treated cells were checked for Sp1 DNA binding activity by EMSA. E, Effect of Berberine on composition of AP-1 in DNA-binding complex. Nuclear proteins of berberine (250 μg/ml) treated cells for different time periods were checked for various AP-1 proteins present in functional AP-1 complex by supershift assay (*Excess of nuclear proteins of 18 h treated cells were used to reveal participation of AP-1 members.) F, Effect of berberine on the expression of AP-1 family proteins. Cellular proteins isolated from berberine (250 μg/ml) treated SiHa cells for indicated time durations were examined for the expression of AP-1 family proteins members. β-actin was used as loading control. The abundance ratio to β-actin was analyzed by densitometry. The data are expressed as the mean ± SD of 3 independent experiments.