Increase in wild-type p53 stability and transactivational activity by the chemopreventive agent apigenin in keratinocytes

Apigenin, a naturally occurring, non-mutagenic flavonoid, has been shown to inhibit UV-induced skin tumorigenesis in mice when topically applied. In t his report we have used the mouse keratinocyte 308 cell line, which contain s a wild-type p53 gene, to study the effect of apigenin treatment on p53 pr otein levels and the expression of its downstream partner, p21/waf1. Cells were treated with 70 mu M apigenin for various times and levels of p53 and p21/waf1 protein were assessed by western blot analysis. The level of p53 p rotein was induced 27-fold after 4 h of apigenin treatment and levels remai ned elevated through 10 h of exposure. After 24 h of exposure to 70 mu M ap igenin, p53 protein levels returned to control levels. p21/waf1 protein lev els increased similar to 1.5-2-fold after 4 h and remained elevated at 24 h . To investigate the mechanism of p53 protein accumulation, me compared the half-life of p53 protein in vehicle- and apigenin-treated cells. Cells wer e incubated for 4 h in the presence of apigenin, then cycloheximide was add ed to inhibit further protein synthesis and p53 protein levels mere measure d by western blot. The half-life of p53 protein was found to be increased a n average of 8-fold in apigenin-treated cells compared with vehicle-treated cells (t 1/2 = 131 min versus 16 min in apigenin- versus vehicle-treated c ells, respectively). The mechanism of p53 protein stabilization is currentl y being investigated. To determine whether p53 was transcriptionally active , me also performed gel mobility shift assays and transient transfection st udies using a luciferase plasmid under the control of the p21/waf1 promoter . Both p53 DNA-binding activity and transcriptional activation peaked after 24 h of exposure to apigenin. These studies suggest that apigenin may exer t anti-tumorigenic activity by stimulating the p53-p21/waf1 response pathwa y.