Effect of artemisinins and other endoperoxides on nitric oxide-related signaling pathway in RAW 264.7 mouse macrophage cells

Abstract

Artemisinin is the active principle of the Chinese herb Artemisia annua L. In addition to its anti-malarial activity, artemisinin and its derivatives have been shown to exert profound anti-cancer activity. The endoperoxide moiety in the chemical structure of artemisinin is thought to be responsible for the bioactivity. Here, we analyzed the cytotoxicity and the ability of artemisinin, five of its derivatives, and two other endoperoxides to inhibit generation of nitric oxide (NO). In the RAW 264.7 mouse macrophage cell line, the well-established model cell line to analyze NO generation, artesunate revealed the highest ability to inhibit NO production among all compounds tested. In cytotoxicity assays (XTT assay), the IC(50) value of RAW 264.7 cells for artesunate was determined to be 3.1+/-0.7 microM. In order to associate the cytotoxic effects with specific alteration in gene expression related to NO metabolism and signaling, whole genome mRNA microarray analyses were conducted. RAW 264.7 cells were treated with artesunate using DMSO as vehicle control followed by microarray analysis. A total of 36 genes related to NO metabolism and signaling were found to be differentially expressed upon exposure to artesunate. Apart from NO-related genes, the expression of genes associated with other functional groups was also analyzed. Out of 24 functional groups, differential expression was most prominent in genes involved in cell-to-cell signaling and interactions. Further refinement of this analysis showed that the pathways for cAMP-mediated signaling and Wnt/beta-catenin signaling were most closely related to changes in mRNA expression. In conclusion, NO generation and signaling play a role in exhibiting cytotoxic activity of artesunate. In addition, other signaling pathways also contribute to the inhibitory effect of artesunate towards RAW 264.7 cells pointing to a multi-factorial mode of action of artesunate.

Artemisinin Analogue SM934 Ameliorates Murine Experimental Autoimmune Encephalomyelitis through Enhancing the Expansion and Functions of Regulatory T Cell

“…Artemisinin and its derivatives, such as Artesunate and Dihydroartemisinin, have been reported to have immune-regulatory effects for many years [37]–[39]. SM934, a water soluble artemisinin analogue, exerted immune-regulatory effects both in vitro and in vivo .…”

Design of novel artemisinin-like derivatives with cytotoxic and anti-angiogenic properties

“…Because artemisinins have been implicated in suppressing angiogenesis via several mechanisms, we utilized the zebrafish embryo model to directly visualize effects on vascular development. We chose the zebrafish due to the availability of a transgenic line that expresses GFP in all endothelial cells [34], which allows direct observation of blood vessel formation. Despite the seemingly distant relationship of human beings and zebrafish, there is a remarkable homology at the genetic level: most human genes have zebrafish orthologues and the zebrafish is more and more recognized as a valuable model for human diseases [62, 63].…”

“…Cytotoxicity was assessed using the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt (XTT) assay kit (Roche, Indianapolis, IN, USA), which measures the metabolic activity of viable cells [33, 34]. Toxicity of compounds was determined with the Cell Proliferation Kit II (Roche Diagnostics, Mannheim, Germany), according to the manufacturer’s instructions.…”

Effects of Scrophularia ningpoensis Hemsl. on Inhibition of Proliferation, Apoptosis Induction and NF-κB Signaling of Immortalized and Cancer Cell Lines

“…The amount of dye is commensurate to the number of metabolic active cells. The procedure has been described by us in detail [24]. …”