Search of cell cycle regulator as an anti-cancer agent, from marine invertebrates

P21最初被鉴定为p53的靶蛋白，然后被发现抑制Cdk-细胞周期蛋白复合物的活性，从而作为刹车调节细胞周期。p21的表达主要受多种机制的调控，且依赖于p53。由于越来越多的证据表明p53在许多人类癌细胞中发生突变，p53的突变已被认为是癌症发生的主要事件之一。然而，似乎p21在人类肿瘤中很少突变。因此，通过p53非依赖性途径诱导p21表达的药物可能有助于癌症的预防或治疗。最近，我们建立了一种使用p53阴性的人骨肉瘤MG 63细胞的生物测定方法，以寻找以p53非依赖性方式激活p21启动子的化合物。在此基础上，我们分离得到了海洋生物碱鱼精蛋白、植物生物碱隐叶碱和细菌聚酮化合物裂酮酸D。Aaptamine在20-50 μg/ml的浓度下激活稳定转染在MG 63细胞中的p21启动子。野生型MG 63细胞中p21的表达也随着鱼精蛋白处理而增加。鱼精蛋白处理MG 63细胞48小时可使细胞周期阻滞于G2/M期。并对aaptamine的p21启动子的响应元件进行了分析。将全长p21启动子和一系列缺失或突变的荧光素酶报告基因融合构建体在MG 63细胞中瞬时表达，并将细胞用aptamine处理，结果表明Sp1-3、Sp1-4和Sp1-5、-6在aptamine激活p21启动子中起重要作用。总之，我们的研究表明，鱼精蛋白通过-82和-50之间的Sp1位点激活p21启动子，从而以不依赖于p53的方式诱导p21表达。

P21 was originally identified as a target protein of p53 and then found to inhibit the activity of Cdk-cyclin complexes, thereby regulating cell cycle as a brake. The p21 expression is mainly controlled by diverse mechanisms in a p53-dependent manner. Due to accumulating evidence that p53 is mutated in many human cancer cells, the mutation of p53 has been recognized as one of the major events in carcinogenesis. However, it appears p21 is rarely mutated in human tumors. Therefore, the agents that induce p21 expression through a p53-independent pathway might contribute to cancer prevention or treatment.Recently, we established a bioassay method using p53-negative human osteosarcoma MG63 cells to search for compounds that activate the p21 promoter in a p53-independent manner. On the guidance of this bioassay, we isolated aaptamine, a marine alkaloid, cryptolepine, a plant alkaloid and secaronic acid D, a bacterial polyketide as active components. Aaptamine activates p21 promoter stably transfected in MG63 cells at the concentrations of 20-50 μg/ml. Expression of p21 in wild-type MG63 cells also increased with aaptamine treatment. The 48 hr treatment of aaptamine induced G2/M arrest of cell cycle in MG63 cells. Furthermore, responsive elements in p21 promote of aaptamine were analyzed. The full length p21 promoter and a series of deleted or mutated constructs fused with luciferase reporter were transiently expressed in MG63 cells, and the cells were treated with aaptamine, showing that Sp1-3, Sp1-4, and Sp1-5, -6 play important roles in the activation of p21 promoter by aaptamine. In conclusion, our investigation indicated that aaptamine activates the p21 promoter through Sp 1 sites between -82 and -50 by and therefore induces p21 expression in a p53-independent manner.