Apigenin is a nonmutagenic bioflavonoid that inhibits UV-induced skin cancer when topically applied to mouse skin and we are currently investigating its molecular mechanism(s) of action. Recent results from our laboratory indicate that inhibition of the cyclooxygenase-2 (COX-2) pathway is one of the ways that apigenin exerts its chemopreventive effect. We have demonstrated that apigenin is a specific inhibitor of UV-induced cyclooxygenase-2 (COX-2) transcription in UV-induced keratinocytes. We have also obtained new evidence since the previous submission that UV-induction of COX-2 expression in keratinocytes requires the E-box and ATF/CRE transcription factor binding sites in the first 200 bases of the COX-2 promoter region, as well as demonstrating a role for the NF-IL6 site. These results suggest that one mechanism by which apigenin inhibits UV-induced skin carcinogenesis is by modulating the function of DNA binding proteins specific for these sites. Recent reports from other laboratories including our collaborator Dr. Aubrey Morrison indicate that COX-2 expression is regulated post-transcriptionally as well, by trans-acting factors which affect COX-2 mRNA stability and modulate translational efficiency. Therefore the hypothesis to be tested in this revised application is that one of the mechanisms by which the bioflavonoid apigenin inhibits UV-induced skin carcinogenesis is through modulation of transcriptional and post-transcriptional control of COX-2 expression. The following aims will test this hypothesis:
In Aim #1 we will continue our efforts to identify the DNA binding proteins in the COX-2 promoter required for UVB-induced COX-2 transcription and for inhibition by apigenin in keratinocytes and mouse epidermis in vivo.
In Aim #2 we will investigate the mechanism by which apigenin treatment of keratinocytes modulates the function of the DNA binding proteins identified in Aim #1.
In Aim #3 we will characterize the post-transcriptional control mechanisms which affect UVB-induced COX-2 expression in keratinocytes treated with apigenin, using regions of the COX-2 3'-UTR to investigate apigenin's ability to interfere with UV-induced mRNA stabilization and/or translational efficiency.
In Aim #4 we will use SKH-1 mice and SKH-1 COX-2 null mice to confirm the ability of apigenin to block UVB-induced COX-2 expression and tumorigenesis in vivo.
