Artemisinin blocks prostate cancer growth and cell cycle progression by disrupting Sp1 interactions with the cyclin-dependent kinase-4 (CDK4) promoter and inhibiting CDK4 gene expression.
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.
Willoughby
Jamin A
JA
Department of Molecular and Cell Biology and Cancer Research Laboratory, University of California at Berkeley, Berkeley, California 94720-3200, USA.
Sundar
Shyam N
SN
Cheung
Mark
M
Tin
Antony S
AS
Modiano
Jaime
J
Firestone
Gary L
GL
D037621:Artemisinins / Q000737:chemistry / Q000494:pharmacology*; D001665:Binding Sites; D002453:Cell Cycle / Q000187:drug effects*; D045744:Cell Line, Tumor; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D051358:Cyclin-Dependent Kinase 4 / Q000235:genetics / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D008297:Male; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation / Q000187:drug effects; D011401:Promoter Regions, Genetic / Q000235:genetics; D011471:Prostatic Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D011485:Protein Binding; D016160:Retinoblastoma Protein / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics
D037621:Artemisinins; D016160:Retinoblastoma Protein; D016329:Sp1 Transcription Factor; C031327:artemisinin; C495900:CDK4 protein, human; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4