1. Jamin A. Willoughby, Sr. ^{‡ §} ,
2. Shyam N. Sundar ^{‡ §} ,
3. Mark Cheung ^{‡ §} ,
4. Antony S. Tin ^{‡ §} ,
5. Jaime Modiano ^{¶ ∥} and
6. Gary L. Firestone ^{‡ § 1}

+ Author Affiliations

1. 
   
   ^‡Department of Molecular and Cell Biology and ^§Cancer Research Laboratory, University of California at Berkeley, Berkeley, California 94720-3200, the ^¶College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota 55108, and the ^∥University of Minnesota Cancer Center, Minneapolis, Minnesota 55455

1. 1 To whom correspondence should be addressed: Dept. of Molecular and Cell Biology, 591 LSA, University of California at Berkeley, Berkeley, CA 94720-3200. Tel.: 510-642-8319; Fax: 510-643-6791; E-mail: glfire@berkeley.edu.

Abstract

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.

Artemisinin and Dog osteosarcoma

Footnotes

• ↵2 The abbreviations used are: CDK, cyclin-dependent kinase; LNCaP, lymph node carcinoma of the prostate; Art, artemisinin; Rb, retinoblastoma protein; pRb, phosphorylation of retinoblastoma protein; ppRb, hyperphosphorylated form of Rb; Sp1, promoter specificity factor; OA, okadaic acid; CKI, cyclin-dependent kinase inhibitor; PBS, phosphate-buffered saline; CMV, cytomegalovirus.

• ↵3 J. A. Willoughby Sr., S. N. Sundar, M. Cheung, A. S. Tin, J. Modiano, and G. L. Firestone, manuscript in preparation.

• ↵* This work was supported, in whole or in part, by National Institutes of Health Grant CA102360 from NCI. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

• • Received June 12, 2008.
  • Revision received October 27, 2008.
• The American Society for Biochemistry and Molecular Biology, Inc.