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 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 E(2) 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 kinase1/2 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.
Subbaramaiah
K
K
Department of Medicine, New York Presbyterian Hospital-Cornell, and Anne Fisher Nutrition Center at Strang Cancer Prevention Center, New York, NY.
Bulic
P
P
Lin
Y
Y
Dannenberg
A J
AJ
Pasco
D S
DS
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D015152:Blotting, Northern; D015153:Blotting, Western; D001940:Breast / Q000473:pathology; D002461:Cell Line, Transformed; D051546:Cyclooxygenase 2; D015232:Dinoprostone / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000235:genetics*; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D008565:Membrane Proteins; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009285:Naphthoquinones / Q000737:chemistry / Q000494:pharmacology; D010936:Plant Extracts / Q000494:pharmacology*; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D000972:Antineoplastic Agents, Phytogenic; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008565:Membrane Proteins; D009285:Naphthoquinones; D010936:Plant Extracts; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; C016101:shikonin; D008156:Luciferases; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D015232:Dinoprostone; D013755:Tetradecanoylphorbol Acetate