Artemisinin triggers a G1 cell cycle arrest of human Ishikawa endometrial cancer cells and inhibits cyclin-dependent kinase-4 promoter activity and expression by disrupting nuclear factor-κB transcriptional signaling.

Relatively little is known about the antiproliferative effects of artemisinin, a naturally occurring antimalarial compound from Artemisia annua, or sweet wormwood, in human endometrial cancer cells. Artemisinin induced a G1 cell cycle arrest in cultured human Ishikawa endometrial cancer cells and downregulated cyclin-dependent kinase-2 (CDK2) and CDK4 transcript and protein levels. Analysis of CDK4 promoter-luciferase reporter constructs showed that the artemisinin ablation of CDK4 gene expression was accounted for by the loss of CDK4 promoter activity. Chromatin immunoprecipitation demonstrated that artemisinin inhibited nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) subunit p65 and p50 interactions with the endogenous Ishikawa cell CDK4 promoter. Coimmunoprecipitation revealed that artemisinin disrupts endogenous p65 and p50 nuclear translocation through increased protein-protein interactions with IκB-α, an NF-κB inhibitor, and disrupts its interaction with the CDK4 promoter, leading to a loss of CDK4 gene expression. Artemisinin treatment stimulated the cellular levels of IκB-α protein without altering the level of IκB-α transcripts. Finally, expression of exogenous p65 resulted in the accumulation of this NF-κB subunit in the nucleus of artemisinin-treated and artemisinin-untreated cells, reversed the artemisinin downregulation of CDK4 protein expression and promoter activity, and prevented the artemisinin-induced G1 cell cycle arrest. Taken together, our results demonstrate that a key event in the artemisinin antiproliferative effects in endometrial cancer cells is the transcriptional downregulation of CDK4 expression by disruption of NF-κB interactions with the CDK4 promoter.

Antiproliferative effects of artemisinin on human breast cancer cells requires the downregulated expression of the E2F1 transcription factor and loss of E2F1-target cell cycle genes.

Artemisinin, a sesquiterpene phytolactone derived from Artemisia annua, is a potent antimalarial compound with promising anticancer properties, although the mechanism of its anticancer signaling is not well understood. Artemisinin inhibited proliferation and induced a strong G1 cell cycle arrest of cultured MCF7 cells, an estrogen-responsive human breast cancer cell line that represents an early-stage cancer phenotype, and effectively inhibited the in-vivo growth of MCF7 cell-derived tumors from xenografts in athymic nude mice. Artemisinin also induced a growth arrest of tumorigenic human breast cancer cell lines with preneoplastic and late stage cancer phenotypes, but failed to arrest the growth of a nontumorigenic human mammary cell line. Concurrent with the cell cycle arrest of MCF7 cells, artemisinin selectively downregulated the transcript and protein levels of the CDK2 and CDK4 cyclin-dependent kinases, cyclin E, cyclin D1, and the E2F1 transcription factor. Analysis of CDK2 promoter-luciferase reporter constructs showed that the artemisinin ablation of CDK2 gene expression was accounted for by the loss of CDK2 promoter activity. Chromatin immunoprecipitation revealed that artemisinin inhibited E2F1 interactions with the endogenous MCF7 cell CDK2 and cyclin E promoters. Moreover, constitutive expression of exogenous E2F1 prevented the artemisinin-induced cell cycle arrest and downregulation of CDK2 and cyclin E gene expression. Taken together, our results demonstrate that the artemisinin disruption of E2F1 transcription factor expression mediates the cell cycle arrest of human breast cancer cells and represents a critical transcriptional pathway by which artemisinin controls human reproductive cancer cell growth.

Indole-3-carbinol downregulation of telomerase gene expression requires the inhibition of estrogen receptor-alpha and Sp1 transcription factor interactions within the hTERT promoter and mediates the G1 cell cycle arrest of human breast cancer cells.

Indole-3-carbinol (I3C), a naturally occurring hydrolysis product of glucobrassicin from cruciferous vegetables such as broccoli, cabbage and Brussels sprouts, is an anticancer phytochemical that triggers complementary sets of antiproliferative pathways to induce a cell cycle arrest of estrogen-responsive MCF7 breast cancer cells. I3C strongly downregulated transcript expression of the catalytic subunit of the human telomerase (hTERT) gene, which correlated with the dose-dependent indole-mediated G(1) cell cycle arrest without altering the transcript levels of the RNA template (hTR) for telomerase elongation. Exogenous expression of hTERT driven by a constitutive promoter prevented the I3C-induced cell cycle arrest and rescued the I3C inhibition of telomerase enzymatic activity and activation of cellular senescence. Time course studies showed that I3C downregulated expression of estrogen receptor-alpha (ERα) and cyclin-dependent kinase-6 transcripts levels (which is regulated through the Sp1 transcription factor) prior to the downregulation of hTERT suggesting a mechanistic link. Chromatin immunoprecipitation assays demonstrated that I3C disrupted endogenous interactions of both ERα and Sp1 with an estrogen response element-Sp1 composite element within the hTERT promoter. I3C inhibited 17ß-estradiol stimulated hTERT expression and stimulated the production of threonine-phosphorylated Sp1, which inhibits Sp1-DNA interactions. Exogenous expression of both ERα and Sp1, but not either alone, in MCF7 cells blocked the I3C-mediated downregulation of hTERT expression. These results demonstrate that I3C disrupts the combined ERα- and Sp1-driven transcription of hTERT gene expression, which plays a significant role in the I3C-induced cell cycle arrest of human breast cancer cells.