Studies on the inhibitors of the catalytic activities and gene expressions of arachidonate oxygenases

花生四烯酸的生物合成是由环氧合酶和脂氧合酶启动的。本研究的目的是寻找这些酶的催化活性的抑制剂和酶基因的转录：1)环氧合酶基因的转录抑制物：早些时候，我们报道了从啤酒啤酒花中分离得到的葫芦酮能抑制环氧合酶-2基因的转录。关于环氧合酶-2在血管生成中的作用，我们发现鸡胚绒毛尿囊膜的血管生成明显地被胡萝卜素抑制。2)12-脂氧合酶抑制剂：早期我们发现，在12-脂氧合酶的三种同工酶：血小板型、白细胞型和表皮型中，Hinokitiol是一种选择性的12-脂氧合酶同工酶。进一步的研究表明，用肉桂醇和肉桂醇的乙醚抑制血小板12-脂氧合酶具有更高的特异性。此外，我们注意到了茶叶儿茶素的抗氧化活性，并测试了它们作为12-脂氧合酶抑制剂的作用。被测试的大多数儿茶素相对选择性地抑制血小板型酶。对(-)-没食子儿茶素没食子酸酯的抑制作用最强，IC50值为0.1μM。此外，约10μM的Hinokitiol对多种黑色素瘤和前列腺癌细胞系的生长有抑制作用。3)前列腺素E2的酶免疫测定：在花生四烯酸的各种环氧合酶代谢产物中，前列腺素E2显示出多种生物活性，其产物被检测为跟随环氧合酶的表达。作为一种简便、灵敏的检测方法，我们尝试建立前列腺素E2的酶免疫检测方法。建立了以乙酰胆碱酯酶为标记物的测定方法，并给出了校正曲线，其IC_(50)为4.5pg/50μL样品。

The biosynthesis of lipid mediators from arachidonic acid is initiated by cyclooxygenase and lipoxygenase enzymes. The purpose of this research project is to search for inhibitors of the catalytic activities of these enzymes and the transcription of enzyme genes.1)Transcription inhibitor of cyclooxygenase gene : Earlier we reported that humulone isolated from beer hop inhibited the transcription of cyclooxygenase-2 gene. In connection of the role of cyclooxygenase-2 in angiogenesis, we found that the angiogenesis of chicken embryo chorioallantoic membrane was markedly inhibited by humulone.2)12-Lipoxygenase inhibitor : Earlier we found that hinokitiol, a tropolon derivative, was a selective inhibitor of the platelet-type among the three isozymes of 12-lipoxygenase ; platelet-, leukocyte- and epidermis-types. Further studies demonstrated a higher specificity of the inhibition of platelet 12-lipoxygenase with an ether of hinokitiol and cinnamic alcohol. Furthermore, we noted the antioxidant activity of tea leaf catechins, and tested them as an inhibitor of 12-lipoxygenase. Most catechins tested inhibited the enzyme of platelet-type relatively selectively. The most potent and selective inhibition was observed with (-)-gallocatechin gallate with an IC50 of 0.1μM. In addition, hinokitiol at about 10μM inhibited the growth of various cell lines of melanoma and prostate cancer3)Enzyme immunoassay of prostaglandin E2 : Among various cyclooxygenase-metabolites of arachidonic acid, prostaglandin E2 shows a variety of biological activities, and its production is assayed to follow the cyclooxygenase expression. As an easy and sensitive assay, we attempted to develop an enzyme immunoassay of prostaglandin E2. We developed an assay with acetylcholine esterase as a label which gave a calibration curve with an IC50 of 4.5 pg/50 μl sample.