Subbaramaiah K, Bulic P, Lin Y, Dannenberg A J, Pasco D S
Department of Medicine, New York Presbyterian Hospital-Cornell, and Anne Fisher Nutrition Center at Strang Cancer Prevention Center, New York, NY.
J Biomol Screen. 2001 Apr;6(2):101-10. doi: 10.1177/108705710100600206.
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.
环氧化酶-2（COX-2）是公认的癌症预防和可能的治疗靶点。为了鉴定新型COX-2抑制剂，我们开发了一种高通量报告基因检测方法，该方法利用人COX-2启动子区域来驱动荧光素酶表达。共筛选了来自266种植物的968份提取物。来自12种植物（4.5%）的提取物，包括在远东地区用于治疗炎症的药用植物新疆紫草，抑制了COX-2启动子活性的刺激。然后，基因启动子检测被用于鉴定紫草素，一种具有已知抗炎和化学预防特性的化合物，作为新疆紫草中的活性化合物。为了补充基因启动子研究，我们测定了紫草素混合物对佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）介导的转化人乳腺上皮细胞中COX-2诱导的影响。紫草素抑制了PMA介导的COX-2 mRNA、蛋白质和前列腺素E2合成的诱导。在瞬时转染中，PMA导致COX-2启动子活性增加数倍，这一效应被紫草素抑制。紫草素还抑制了PMA介导的细胞外信号调节激酶1/2丝裂原活化蛋白激酶和活化蛋白-1活性的刺激。总的来说，这些结果证明了高通量报告基因检测方法在鉴定新型COX-2表达抑制剂方面的成功开发和应用。
