Induction of p21/WAF1 and G1 cell-cycle arrest by the chemopreventive agent apigenin

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Abstract
Apigenin is a plant flavonoid that has been shown to significantly inhibit ultraviolet‐induced mouse skin tumorigenesis when applied topically and may be an alternative sunscreen agent for humans. A long‐term goal of our laboratory is to elucidate the molecular mechanism or mechanisms by which apigenin inhibits skin tumorigenesis. In a previous publication, we characterized the mechanism by which apigenin induced G2/M arrest in keratinocytes. More recent studies in our laboratory have provided evidence that apigenin can induce G1 arrest in addition to arresting cells at G2/M. Here we describe the mechanism of the apigenin‐induced G1 arrest in human diploid fibroblasts (HDF). Treatment of asynchronous HDF for 24 h with 10–50 μM apigenin resulted in dose‐dependent cell‐cycle arrest at both the G0/G1 and G2/M phases as measured by flow cytometry. The G0/G1 arrest was more clearly defined by using HDF that were synchronized in G0 and then released from quiescence by replating at subconfluent densities in medium containing 10–70 μM apigenin. The cells were analyzed for cell‐cycle progression or cyclin D1 expression 24 h later. A dose of apigenin as low as 10 μM reduced the percentage of cells in S phase by 20% compared with control cultures treated with solvent alone. Western blot analysis of apigenin‐treated HDF indicated that cyclin D1 was expressed at higher levels than in untreated cells, which signifies that they were arrested in G1 phase rather than in a G0 quiescent state. The G1 arrest was further studied by cyclin‐dependent kinase 2 (cdk2) immune complex–kinase assays of apigenin‐treated asynchronous HDF, which demonstrated a dose‐dependent inhibition of cdk2 by apigenin. Inhibition of cdk2 kiase activity in apigenin‐treated cells was associated with the accumulation of the hypophosphorylated form of the retinoblastoma (Rb) protein as measured by western blot analysis. The cdk inhibitor p21/WAF1 was also induced in a dose‐dependent manner, with a 22‐fold induction of p21/WAF1 in 70 μM apigenin‐treated cells. In conclusion, apigenin treatment produced a G1 cell‐cycle arrest by inhibiting cdk2 kinase activity and the phosphorylation of Rb and inducing the cdk inhibitor p21/WAF1, all of which may mediate its chemopreventive activities in vivo. To our knowledge this is the first report of a chemopreventive agent inducing p21/WAF1, a known downstream effector of the p53 tumor suppressor protein. Mol. Carcinog. 19:74–82, 1997.