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

Department of Molecular and Cell Biology and Cancer Research Laboratory, University of California at Berkeley, Berkeley, California 94720-3200, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 12/2008; 284(4):2203-13. DOI: 10.1074/jbc.M804491200
Source: PubMed


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.

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Available from: Antony S Tin, Aug 04, 2015
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    • "While this is the first genomics scale study suggesting a role for Sp1 in SFN induced chemoprevention, there is a growing body of evidence that supports Sp1 as a target of natural chemopreventive phytochemicals in cancer cells [32]. Treatment of prostate cancer cells with the natural products epigallocatechin gallate, betulinic acid, artemisinin, and phenethyl isothiocyanate is also associated with inhibition of Sp1 activity and/or expression [40] [41] [42] [43]. In these studies, inhibition of Sp1 is associated with inhibition of the cancer promoting genes surviving, CDK4, VEGF and the androgen receptor. "
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    ABSTRACT: Scope: Epidemiological studies provide evidence that consumption of cruciferous vegetables, like broccoli, can reduce the risk of cancer development. Sulforaphane (SFN) is a phytochemical derived from cruciferous vegetables that induces anti-proliferative and pro-apoptotic responses in prostate cancer cells, but not in normal prostate cells. The mechanisms responsible for this cancer-specific cytotoxicity remain unclear. Methods and results: We utilized RNA sequencing and determined the transcriptomes of normal prostate epithelial cells, androgen-dependent prostate cancer cells, and androgen-independent prostate cancer cells treated with SFN. SFN treatment dynamically altered gene expression and resulted in distinct transcriptome profiles depending on prostate cell line. SFN also down-regulated the expression of genes that were up-regulated in prostate cancer cells. Network analysis of genes altered by SFN treatment revealed that the transcription factor Specificity protein 1 (Sp1) was present in an average of 90.5% of networks. Sp1 protein was significantly decreased by SFN treatment in prostate cancer cells and Sp1 may be an important mediator of SFN-induced changes in expression. Conclusion: Overall, the data show that SFN alters gene expression differentially in normal and cancer cells with key targets in chemopreventive processes, making it a promising dietary anti-cancer agent.
    Molecular Nutrition & Food Research 10/2014; 58(10). DOI:10.1002/mnfr.201400269 · 4.60 Impact Factor
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    • "Cyclins promote S phase, whereas p27 and p21 maintain cells in G1 arrest. CDK4, p27 and p21 have been reported to regulate the G1/S-phase transition and are potentially regulated by Sp1 [35]–[37]. To study whether Sp1 promotes cell cycle through the expression regulation of these molecules in nasopharyngeal carcinoma, CNE2, HNE1 and HK1 cells were transfected with siSp1 and siNC for 48 h, followed by analysis for the levels of Sp1, CDK4, p27 and p21. "
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    ABSTRACT: Background Transcription factor Sp1 is multifaceted, with the ability to function as an oncogene or a tumor suppressor, depending on the cellular context. We previously reported that Sp1 is required for the transcriptional activation of the key oncogenes in nasopharyngeal carcinoma (NPC), including B-lymphoma mouse Moloney leukemia virus insertion region 1 (Bmi1) and centromere protein H (CENPH), but the role of Sp1 and its underlying mechanisms in NPC remained largely unexplored. The objective of this study was to investigate the cellular function of Sp1 and to verify the clinical significance of Sp1 as a potential therapeutic target in NPC.Methods The levels of Sp1 in the normal primary nasopharyngeal epithelial cells (NPECs) and NPC cell lines were analyzed by Quantitative Real-time RT-PCR (qRT-PCR) and Western blot. The location and expression of Sp1 in the NPC tissues were detected by immunohistochemistry staining (IHC). The effect of Sp1 knockdown on the cell proliferation, clonogenicity, anchorage-independent growth and the stem-cell like phenotype in NPC cells were evaluated by MTT, flow cytometry, clonogenicity analysis and sphere formation assay.ResultsThe mRNA and protein levels of Sp1 were elevated in NPC cell lines than in the normal primary NPECs. Higher expression of Sp1 was found in NPC tissues with advanced clinical stage (P¿=¿0.00036). Either inhibition of Sp1 activity by mithramycin A, the FDA-approved chemotherapeutic anticancer drug or Sp1 silencing by two distinct siRNA against Sp1 suppressed the growth of NPC cells. Mechanism analysis revealed that Sp1 silencing may suppress cell proliferation, clonogenicity, anchorage-independent growth and the stem-cell like phenotype through inducing the expression of p27 and p21, and impairing the expressions of the critical stem cell transcription factors (SCTFs), including Bmi1, c-Myc and KLF4 in NPC cells.Conclusions Sp1 was enriched in advanced NPC tissues and silencing of Sp1 significantly inhibited cell proliferation, clonogenicity, anchorage-independent growth and the stem-cell like phenotype of NPC cells, suggesting Sp1 may serve as an appealing drug target for NPC.
    Journal of Translational Medicine 08/2014; 12(1):222. DOI:10.1186/s12967-014-0222-1 · 3.93 Impact Factor
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    • "However, when used as a single agent, HDACi might exhibit limited lethal activity towards liver cancer, which is evident in the present study showing that both NaB and SAHA at low doses are unable to induce significant growth inhibition in vitro and in vivo. However, when HDACi are used in combination with DHA, a derivative of artemisinin that is clinically used in malaria treatment with good toxicity profile [26], they can induce much more apoptosis, resulting in remarkable halt of tumor xenograft in nude mice. There is very limited information on HDACi in combination with other anti-tumor agents against liver cancer. "
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    ABSTRACT: Liver cancer ranks in prevalence and mortality among top five cancers worldwide. Accumulating interests have been focused in developing new strategies for liver cancer treatment. We have previously showed that dihydroartemisinin (DHA) exhibited antitumor activity towards liver cancer. In this study, we demonstrated that histone deacetylase inhibitors (HDACi) significantly augmented the antineoplastic effect of DHA via increasing apoptosis in vitro and in vivo. Inhibition of ERK phosphorylation contributed to DHA-induced apoptosis, due to the fact that inhibitor of ERK phosphorylation (PD98059) increased DHA-induced apoptosis. Compared with DHA alone, the combined treatment with DHA and HDACi reduced mitochondria membrane potential, released cytochrome c into cytoplasm, increased p53 and Bak, decreased Mcl-1 and p-ERK, activated caspase 3 and PARP, and induced apoptotic cells. Furthermore, we showed that HDACi pretreatment facilitated DHA-induced apoptosis. In Hep G2-xenograft carrying nude mice, the intraperitoneal injection of DHA and SAHA resulted in significant inhibition of xenograft tumors. Results of TUNEL and H&E staining showed more apoptosis induced by combined treatment. Immunohistochemistry data revealed the activation of PARP, and the decrease of Ki-67, p-ERK and Mcl-1. Taken together, our data suggest that the combination of HDACi and DHA offers an antitumor effect on liver cancer, and this combination treatment should be considered as a promising strategy for chemotherapy.
    PLoS ONE 06/2012; 7(6):e39870. DOI:10.1371/journal.pone.0039870 · 3.23 Impact Factor
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