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Development and Use of a Gene Promoter-Based Screen to Identify Novel Inhibitors of Cyclooxygenase-2 Transcription

Department of Medicine, New York Presbyterian Hospital-Cornell, and Anne Fisher Nutrition Center at Strang Cancer Prevention Center, New York, NY

Predrag Bulic

National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, University, MS

Yuan Lin

Marco Polo Technologies Inc., Bethesda, MD

Andrew J. Dannenberg

Department of Medicine, New York Presbyterian Hospital-Cornell, and Anne Fisher Nutrition Center at Strang Cancer Prevention Center, New York, NY

David S. Pasco

Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, University, MS

Cyclooxygenase-2 (COX-2) is a recognized target for cancer prevention and possibly treatment. To identify novel inhibitors of COX-2, we developed a high 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 euchroma, a medicinal plant used in the Far East to treat inflammation, inhibited the stimulation of COX-2 promoter activity. The gene promoter assay then was used to identify shikonin, a compound with known anti-inflammatory and chemopreventive properties, as an active compound in A. euchroma. To complement the gene promoter studies, we determined the effects of a mixture of shikonins on phorbol 12-myristate 13-acetate (PMA)-mediated induction of COX-2 in transformed human mammary epithelial cells. Shikonins inhibited PMA-mediated induction of COX-2 mRNA, protein, and prostaglandin E₂ synthesis. In transient transfections, PMA caused a severalfold increase in COX-2 promoter activity, an effect that was suppressed by shikonins. Shikonins also inhibited PMA-mediated stimulation of extracellular signal-regulated kinasel/2 mitogen-activated protein kinases and activator protein-1 activity. Collectively, these results demonstrate the successful development and use of a high throughput reporter gene assay for the identification of a novel inhibitor of COX-2 expression.

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