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

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 mechanism 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 microM 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 microM apigenin. The cells were analyzed for cell-cycle progression or cyclin D1 expression 24 h later. A dose of apigenin as low as 10 microM 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 kinase 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 microM 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.

Mitogen-induced Rapid Phosphorylation of Serine 795 of the Retinoblastoma Gene Product in Vascular Smooth Muscle Cells Involves ERK Activation

“…Fifth, evidence for a role of the Ras 3 Raf 3 MEK 3 ERK pathway in the regulation of the cell cycle was provided by the use of apigenin, a plant flavonoid. In Ras-transformed cells, apigenin reverses the transformed phenotype, diminishes ERK activity and expression of the oncogenes c-fos and c-jun (33), and arrests the cell cycle at the G 0 -G 1 interface (31). Apigenin also inhibited the activity of CDK2 kinase and increased the levels of hypophosphorylated Rb in human diploid fibroblasts (31).…”

Apigenin and cancer chemoprevention: Progress, potential and promise (Review)

“…Topical application of apigenin has been shown to inhibit UV-mediated induction of ornithine decarboxylase activity, reduce tumor incidence and increase tumor-free survival in mice. Several other studies have provided evidence that apigenin prevents UV-induced skin tumorigenesis by inhibiting the cell cycle and cyclin-dependent kinases (51). Exposure of mouse keratinocytes to apigenin induced G2/M cell cycle arrest and accumulation of the p53 tumor suppressor protein with increased expression of p21/WAF1.…”

“…It has been shown that exposure of a wide array of malignant cells, including epidermal cells and fibroblasts to apigenin induces a reversible G2/M and G0/G1 arrest by inhibiting p34 (cdc2) kinase activity, accompanied by increased p53 protein stability (50,51). Apigenin has also been shown to induce WAF1/p21 levels resulting in cell cycle arrest and apoptosis in androgen-responsive human prostate cancer, LNCaP cells and androgen-refractory DU145 cells, regardless of the Rb status and p53-dependence or p53 independence (52,53).…”

Bioactive substances of plant origin in food - impact on genomics

“…According to recent advances in the interpretation of events that occur during programmed cell death, the generation and spreading of ROS within the cell are consequences of the increased permeability of the mitochondrial membrane [80,109]. Lepley and Pelling [57] during a novel cell culture study with apigenin (a quercetin derivative), obtained evidence that flavonoid antioxidants may enhance apoptosis in certain tumor cell lines. Hydrogen peroxide (H 2 O 2 ), similarly to quercetin has been reported to stimulate the activity of the mitogen-activated protein kinases (MAPKs) ERK and JNK, and the expression of the proto-oncogenes c-fos and c-jun [97].…”