Willoughby Jamin A, Sundar Shyam N, Cheung Mark, Tin Antony S, Modiano Jaime, Firestone Gary L
Department of Molecular and Cell Biology and Cancer Research Laboratory, University of California at Berkeley, Berkeley, California 94720-3200, USA.
J Biol Chem. 2009 Jan 23;284(4):2203-13. doi: 10.1074/jbc.M804491200. Epub 2008 Nov 17.
Artemisinin, a naturally occurring component of Artemisia annua, or sweet wormwood, is a potent anti-malaria compound that has recently been shown to have anti-proliferative effects on a number of human cancer cell types, although little is know about the molecular mechanisms of this response. We have observed that artemisinin treatment triggers a stringent G1 cell cycle arrest of LNCaP (lymph node carcinoma of the prostate) human prostate cancer cells that is accompanied by a rapid down-regulation of CDK2 and CDK4 protein and transcript levels. Transient transfection with promoter-linked luciferase reporter plasmids revealed that artemisinin strongly inhibits CDK2 and CDK4 promoter activity. Deletion analysis of the CDK4 promoter revealed a 231-bp artemisinin-responsive region between -1737 and -1506. Site-specific mutations revealed that the Sp1 site at -1531 was necessary for artemisinin responsiveness in the context of the CDK4 promoter. DNA binding assays as well as chromatin immunoprecipitation assays demonstrated that this Sp1-binding site in the CDK4 promoter forms a specific artemisinin-responsive DNA-protein complex that contains the Sp1 transcription factor. Artemisinin reduced phosphorylation of Sp1, and when dephosphorylation of Sp1 was inhibited by treatment of cells with the phosphatase inhibitor okadaic acid, the ability of artemisinin to down-regulate Sp1 interactions with the CDK4 promoter was ablated, rendering the CDK4 promoter unresponsive to artemisinin. Finally, overexpression of Sp1 mostly reversed the artemisinin down-regulation of CDK4 promoter activity and partially reversed the cell cycle arrest. Taken together, our results demonstrate that a key event in the artemisinin anti-proliferative effects in prostate cancer cells is the transcriptional down-regulation of CDK4 expression by disruption of Sp1 interactions with the CDK4 promoter.
青蒿素是黄花蒿（即甜艾）中的一种天然成分，是一种强效抗疟疾化合物，最近已被证明对多种人类癌细胞类型具有抗增殖作用，尽管对这种反应的分子机制了解甚少。我们观察到，青蒿素处理会引发LNCaP（前列腺淋巴结癌）人前列腺癌细胞的严格G1期细胞周期停滞，同时伴随着CDK2和CDK4蛋白及转录水平的快速下调。用启动子连接的荧光素酶报告质粒进行瞬时转染表明，青蒿素强烈抑制CDK2和CDK4启动子活性。对CDK4启动子的缺失分析揭示了-1737至-1506之间有一个231bp的青蒿素反应区域。位点特异性突变表明，在CDK4启动子的背景下，-1531处的Sp1位点对于青蒿素反应是必需的。DNA结合试验以及染色质免疫沉淀试验表明，CDK4启动子中的这个Sp1结合位点形成了一个特定的青蒿素反应性DNA-蛋白质复合物，其中包含Sp1转录因子。青蒿素降低了Sp1的磷酸化，当用磷酸酶抑制剂冈田酸处理细胞抑制Sp1的去磷酸化时，青蒿素下调Sp1与CDK4启动子相互作用的能力被消除，导致CDK4启动子对青蒿素无反应。最后，Sp1的过表达大多逆转了青蒿素对CDK4启动子活性的下调，并部分逆转了细胞周期停滞。综上所述，我们的结果表明，青蒿素在前列腺癌细胞中抗增殖作用的一个关键事件是通过破坏Sp1与CDK4启动子的相互作用来转录下调CDK4的表达。
