Inhibition of UVB-induced COX-2 expression by apigenin

描述(申请人提供)：芹菜素是一种非诱变生物黄酮类化合物，局部应用于小鼠皮肤可抑制紫外线诱发的皮肤癌，我们目前正在研究其作用的分子机制(S)。本实验室最近的研究结果表明，抑制环氧合酶-2(COX-2)途径是芹菜素发挥化学预防作用的途径之一。我们已经证明，芹菜素是紫外线诱导的角质形成细胞中环氧合酶-2(COX-2)转录的特异性抑制物。自从上一篇论文发表以来，我们还获得了新的证据，即紫外线诱导角质形成细胞中COX-2的表达需要COX-2启动子区域前200个碱基上的E-box和ATF/Cre转录因子结合位点，以及NF-IL6位点的作用。这些结果表明，芹菜素抑制紫外线诱导的皮肤癌的一个机制是通过调节这些部位特异的DNA结合蛋白的功能。最近，包括我们的合作者奥布里·莫里森博士在内的其他实验室的报告表明，COX-2的表达也在转录后受到影响COX-2 mRNA稳定性和调节翻译效率的反式作用因子的调节。因此，在这一修订的应用中要检验的假设是，生物黄酮类芹菜素抑制紫外线诱导的皮肤癌的机制之一是通过调控COX-2的转录和转录后调控。以下目标将验证这一假设：在目标1中，我们将继续努力鉴定体内中UVB诱导的COX-2转录所需的COX-2启动子中的DNA结合蛋白以及芹菜素对角质形成细胞和小鼠表皮的抑制作用。在目标#2中，我们将研究芹菜素处理角质形成细胞调节在目标#1中确定的DNA结合蛋白的功能的机制。在目标#3中，我们将描述影响UVB诱导的角质形成细胞中COX-2表达的转录后调控机制，利用COX-2 3‘-UTR区域来研究芹菜素干扰UV诱导的mRNA稳定和/或翻译效率的能力。在目标4中，我们将使用SKH-1小鼠和SKH-1 COX-2缺失小鼠来证实芹菜素在体内阻断UVB诱导的COX-2表达和肿瘤形成的能力。

DESCRIPTION (provided by applicant): 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 #1we 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.