Human neuroblastoma cell lines SK-N-AS, SK-N-DZ and SHEP1 were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics penicillin and streptomycin (P/S). BE(2)-C cells were cultured in a 1:1 mixture of DMEM and Ham’s nutrient mixture F12 (DMEM/F12), supplemented with 10% FBS and 1% antibiotics (P/S). All four cell lines were purchased from ATCC (Manassas, VA, USA). The growth media, antibiotics and FBS were purchased from Life Technologies. All cells were cultured at 37°C in a 5% CO₂ humidified incubator.

Artemisinin was dissolved in DMSO. The cell growth curve was detected using the Cell Counting Kit-8 assay (CCK-8; Beyotime, China). Briefly, approximately 1,000 cells in 200 μl medium were seeded in 96-well plates and incubated with artemisinin at concentrations of 100, 200, 300 and 400 μM; DMSO was used as a control. CCK-8 (20 μl) was added and incubated for 2 h every 2 days, and the absorbance at 450 nm was used to detect metabolically intact cells according to the manufacturer’s protocol. Cells were exposed to 300 μM artemisinin or DMSO for 72 h, and cell morphologic examination was carried out using an inverted microscope (TS100, Nikon). Then adherent cells were collected and washed with ice-cold PBS. The sample obtained was analyzed by the TC10™ Automated Cell Counter (Bio-Rad, Hercules, CA, USA).

Cells were grown on coverslips and treated with either DMSO or 300 μM artemisinin for 72 h. Cells were washed with PBS, fixed for 20 min in 4% paraformaldehyde (PFA), and permeabilized with 0.3% Triton X-100 for 5 min. The cells were blocked with 10% goat serum in PBS for 1 h, incubated with a primary rat antibody against Ki67 (1:300, 556003; BD) in blocking buffer for 1 h at room temperature, and then incubated with the secondary antibody Alexa Fluor 595 goat anti-mouse IgG (H+L) (1:2,000; A-21422; Invitrogen). DAPI (300 nM) in PBS was used for nuclear staining. Cells were examined using a Nikon microscope (80i) with Image-Pro Plus software for image analysis. Ki67 uptake was calculated in 10 microscopic fields.

Cells were plated in 10-cm plates and treated either with 300 μM artemisinin or DMSO. After 72 h of treatment, cells were fixed with 70% cold ethanol, stained with propidium iodide (PI) and analyzed by flow cytometry (Accuri C6; BD). The data were analyzed with ModfitLT software.

Cells treated with artemisinin for 72 h were harvested and suspended in RIPA lysis buffer (Beyotime, China). Protein concentrations were determined with the Enhanced BCA protein assay kit (Beyotime). Sixty micrograms of protein was separated on 10% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE), transferred to PVDF membranes, and analysis was performed with the primary antibodies including anti-cyclinD1 (1:500; Santa Cruz), anti-α-tubulin (1:2,000; Sigma-Aldrich), anti-CDK4 (1:500; Santa Cruz), anti-cyclinE2 (1:500; Santa Cruz), anti-cyclinB1 (1:500; Santa Cruz). Horseradish peroxidase-conjugated goat anti-mouse (1:20,000) and rabbit anti-goat (1:10,000) immunoglobulin G (IgG; KPL, Gaithersburg, MD, USA) were used as secondary antibodies. Proteins were visualized with BeyoECL Plus (Beyotime, China).

For cell death assay, all cells grown to 60–70% confluency were treated with 300 μM artemisinin for 72 h, and cells were treated with DMSO as control. After treatment, adherent and floating cells were collected by centrifugation, and then the sample obtained was analyzed using the TC10™ Automated Cell Counter. Cell counting was carried out using trypan blue dye (145-0021, Bio-Rad) staining as previously described (22). The apoptotic ratios of the cells were determined with the Annexin V-FITC apoptosis detection kit (Sigma). Briefly, after a 72-h treatment with DMSO or 300 μM artemisinin, the cells were collected and washed twice with cold PBS buffer, resuspended in 100 μl of binding buffer, and incubated with 5 μl of Annexin V conjugated to FITC and 10 μl PI for 15 min at room temperature. Cell were then analyzed by flow cytometry (Accuri C6; BD). The data were analyzed with Flowjo software.

Five hundred cells were mixed with 0.3% Noble agar in growth medium and plated into 6-well plates containing a solidified bottom layer (0.6% Noble agar in growth medium). After 21 days of cell growth, colonies were stained with 5 mg/ml MTT (Sigma), photographed and recorded.

BE(2)-C cells were grown to 70–80% confluency and trypsinized. Cells (1×10⁶ in 200 μl DMEM) were injected into the flanks of NOD/SCID mice. After one week of tumor growth, the mice were administered intraperitoneal injections of artemisinin at 100 mg/kg (mouse body weight) daily or vehicle control DMSO (1 μl/ml DMEM) (16) for 12 days. Tumor diameter was measured with digital calipers every 3 days, and the tumor volume (V) was calculated by the formula: V = 1/2 (length × width²). After treatment, mice were sacrificed by CO₂, and tumors were observed and weighed. All animal experiments were pre-approved by the Institutional Animal Care and Use Committee of Chongqing University.

Quantitative data are expressed as the mean ± SD. The two-tailed Student’s t-test was performed for paired samples. A minimum of three independent experiments was performed. Differences were considered statistically significant at P<0.05.

Neuroblastoma cells were treated with various concentrations of artemisinin, from 100 μM to 400 μM, for an indicated period of time. As shown in Fig. 1A, a concentration- and time-dependent response to artemisinin in neuroblastoma cells was observed. Artemisinin markedly inhibited cell proliferation in the neuroblastoma cells. After 72 h of treatment with 300 μM artemisinin, both morphologic examination and cell counting revealed that artemisinin significantly reduced cell growth in all four neuroblastoma cell lines (Fig. 1B and C). As a proliferation marker, Ki67 staining also confirmed that artemisinin markedly inhibited cell proliferation (Fig. 2A). The histogram statistics of the Ki67-positive rates also demonstrated that artemisinin was an effective agent for inhibiting the proliferation of neuroblastoma cells (Fig. 2B). The percentage of Ki67-positive SK-N-AS cells was reduced from 67.2±2.76 to 25.2±6.11%. The percentage of Ki67-positive BE(2)-C cells was reduced from 72.8±4.94 to 22.7±1.51%. The percentage of Ki67-positive SK-N-DZ cells was reduced from 82.1±4.89 to 41.1±5.08%, and the percentage of Ki67-positive SHEP1 cells was reduced from 57.6±1.88 to 37.9±4.85%.

Furthermore, to gain insight into artemisinin-induced inhibition of cell proliferation, we examined the cell cycle distribution of the four neuroblastoma cell lines. After artemisinin treatment at 300 μM for 72 h, the proportion of cells in the G1 phase was significantly increased in all four cell lines (Fig. 3A). The cell cycle analysis of artemisinin-treated BE(2)-C cells revealed a significant increase in the proportion of cells in the G0/G1 phase (54.46±2.14%), compared with the vehicle-treated cells (G0/G1, 37.53±1.82%). Similar results were obtained in the SK-N-AS, SK-N-DZ and SHEP1 cell lines (Fig. 3B). These data demonstrated that artemisinin induced cell cycle arrest in the G1 phase. Western blot analysis also showed that artemisinin led to a marked downregulation of cyclinD1, CDK4 and cyclinE2 (Fig. 3C), which are collectively required for cell cycle progression through the G1 to S phases (23,24). These data suggest that artemisinin inhibits cell proliferation through cell cycle arrest in neuroblastoma cells.

To investigate whether artemisinin induces apoptosis in neuroblastoma cells, SK-N-AS, BE(2)-C, SK-N-DZ, and SHEP1 cell lines were incubated with 300 μM artemisinin for 72 h. Trypan blue assay indicated that the cell death was increased after artemisinin incubation when compared with the control (Fig. 4A). Notably, treatment with artemisinin resulted in a higher proportion of cells with positive Annexin V and/or PI staining. Artemisinin markedly induced apoptosis in all four neuroblastoma cell lines (Fig. 4B), which indicated that artemisinin induced cell death through apoptosis in neuroblastoma cells.

Soft agar is used to test the ability of single cancer cells to proliferate and form colonies, and is also used as a ‘human tumor stem-cell assay’ (25). In the present study, BE(2)-C cells treated with 300 μM ART were observed to give rise to small and scant colonies in soft agar compared with the cells treated with DMSO (Fig. 5A and B). To determine the effect of artemisinin on tumorigenicity in neuroblastoma cells, we carried out a xenograft study in NOD/SCID mice. BE(2)-C cells were implanted subcutaneously into the flanks of NOD/SCID mice. One week after tumor injection, mice were treated intraperitoneally with either DMSO or artemisinin at 100 mg/kg daily for 12 days. The volume and weight of the xenograft tumors in the artemisinin treatment group were much smaller and lighter than those in the DMSO group (Fig. 5C and D). These data indicate that artemisinin significantly suppresses the tumorigenicity of neuroblastoma cells.