The Effects of Artemisinin on the Cytolytic Activity of Natural Killer (NK) Cells

Artemisinin, a chemical compound used for the treatment of malaria, has been known to show anti-cancer activity. However, the effect of this chemical on natural killer (NK) cells, which are involved in tumor killing, remains unknown. Here, we demonstrate that artemisinin exerts a potent anti-cancer effect by activating NK cells. NK-92MI cells pre-treated with artemisinin were subjected to a cytotoxicity assay using K562 cells. The results showed that artemisinin significantly enhances the cytolytic activity of NK cells in a dose-dependent manner. Additionally, the artemisinin-enhanced cytotoxic effect of NK-92MI cells on tumor cells was accompanied by the stimulation of granule exocytosis, as evidenced by the detection of CD107a expression in NK cells. Moreover, this enhancement of cytotoxicity by artemisinin was also observed in human primary NK cells from peripheral blood. Our results suggest that artemisinin enhances human NK cell cytotoxicity and degranulation. This is the first evidence that artemisinin exerts antitumor activity by enhancing NK cytotoxicity. Therefore, these results provide a deeper understanding of the action of artemisinin and will contribute to the development and application of this class of compounds in cancer treatment strategies.

as templates for PCR, with primers to detect the RNA levels of granzyme A, granzyme B, perforin, and β-actin. (b) NK92-MI cells treated with 0.1 μM artemisinin (red line) or left untreated (black line), for 48 hours were stained with antibodies to detect intracellular protein levels of perforin and granzyme A by flowcytometry. (c) NK92-MI cells were treated with 0.1 μM artemisinin for 24, or 48 hours. Protein levels of granzyme B were detected by western blot assay. The data shown are representative of three independent experiments. Figure S4. NK92-MI cells treated with 0.1 μM artemisinin for 48 hours were stained with FITCconjugated-mouse anti-human CD2 or CD11a antibodies for 30 minutes on ice. After washing with PBS, the fluorescence intensity was detected by flow cytometry through an FL-1 filter. The data shown are representative of three independent experiments.

Flow cytometry
To confirm whether artemisinin influences the expression of adhesion receptors, CD2 and CD11a were detected using FITC-conjugated antibodies (BD Bioscience). NK92-MI cells were treated with 0.1 μM artemisinin, or left untreated, for 48 hours, and then the cells were incubated with mouse anti-human CD2 or CD11a antibodies for 30 minutes on ice. To confirm the levels of proteins present in intracellular granules, cells were fixed and permeabilized with reagents (BD Bioscience), and then incubated with mouse antibodies against human perforin or granzyme A for 30 minutes on ice. FITC-conjugated goat anti-mouse IgG was used as the secondary antibody (BD Bioscience). After washing, the expression levels of adhesion receptors and intracellular granules were detected using FACSCalibur (Becton Dickinson), with data analysis using FlowJo software.

Western blotting
Cells were collected and washed with PBS. Cells were lysed in lysis buffer (Cell Signaling Technology) containing PMSF and phosphatase inhibitor cocktail on ice. Equal amounts of proteins were loaded on 12% SDS-PAGE, and then transferred onto a PVDF membrane (Bio-Rad). Blocking solution containing 5% non-fat dried milk (Santa Cruz Biotechnology, Inc) was used to block the membrane for 30 minutes.
NK-92MI cells were treated with DMSO used as a vehicle control for 5, 10, or 15 minutes were loaded and rabbit anti-human antibodies were used to detect total ERK 1/2 and phosphorylated ERK 1/2 (Cell Signaling Technology) After washing with PBS containing 0.1% Tween-20, the membrane was stained with goat anti-rabbit IgG peroxidase (Jackson ImmunoResearch Laboratories).
To confirm the expression of granzyme B, NK-92MI cells were treated with 100 μM artemisinin for 24, or 48 hours. Ten micrograms of protein from each sample were loaded, and a polyclonal rabbit anti-human granzyme B antibody (Cell Signaling Technology) was used to detect granzyme B levels.
All the proteins used for western blotting were detected using Amersham ECL Western blotting detection reagent (GE Healthcare) and signals were detected using a chemiluminescenceimaging device LAS-3000 (Fujifilm).