Development and use of a gene promoter-based screen to identify novel inhibitors of cyclooxygenase-2 transcription

Cyclooxygenase-2 (COX-2) is a recognized target for cancer prevention and p ossibly treatment. To identify novel inhibitors of COX-2, we developed a hi gh throughput reporter gene assay that utilizes a region of the human COX-2 promoter to drive luciferase expression. A total of 968 extracts from 266 plants were screened. Extracts from 12 plants (4.5 %), including Arnebia eu chroma, a medicinal plant used in the Far East to treat inflammation, inhib ited the stimulation of COX-2 promoter activity. The gene promoter assay th en was used to identify shikonin, a compound with known anti-inflammatory a nd chemopreventive properties, as an active compound in A. euchroma. To com plement the gene promoter studies, we determined the effects of a mixture o f shikonins on phorbol 12-myristate 13-acetate (PMA)-mediated induction of COX-2 in transformed human mammary epithelial cells. Shikonins inhibited PM A-mediated induction of COX-2 mRNA, protein, and prostaglandin E-2 synthesi s. In transient transfections, PMA caused a severalfold increase in COX-2 p romoter activity, an effect that was suppressed by shikonins. Shikonins als o inhibited PMA-mediated stimulation of extracellular signal-regulated kina se1/2 mitogen-activated protein kinases and activator protein-1 activity. C ollectively, these results demonstrate the successful development and use o f a high throughput reporter gene assay for the identification of a novel i nhibitor of COX-2 expression.