Shikonin and dimethylsulfoxide were purchased from Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The purity of shikonin was >98%. Antibodies against caspase-3, poly ADP-ribose polymerase (PARP), c-Myc, p-ERK1/2, ERK1/2, p38 MAPK, p-JNK and JNK were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The antibody against p-p38 MAPK was purchased from Merck KGaA. Goat anti-rabbit, goat anti-mouse and β-actin antibodies were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. (Beijing, China).

NB4 cells were obtained from the Shanghai Institute for Biological Science, Chinese Academy of Sciences (Shanghai, China) and maintained at 37°C under 5% CO₂ in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) medium containing 10% fetal calf serum (Gibco; Thermo Fisher Scientific, Inc.).

Cell viability was detected by the Cell Counting Kit (CCK)-8 (Sevenseas Futai Biotechnology Co., Ltd., Shanghai, China) assay. Cells (1×10⁴) were seeded in 96-well plates and treated with increasing concentrations of shikonin for 12, 24 and 36 h. A total of 10 µl CCK-8 solution was added to each well at the end of each culture period. Cells were then incubated for 2 h at 37°C and absorbance of the medium was measured at 450 nm using a spectrophotometer.

Cells (5×10⁵) were seeded in six-well plates and treated with shikonin (0, 0.3 µmol/l) for 24 h. Then, cells were washed with PBS three times, fixed with cold methanol overnight. Cells were washed with PBS three further times and stained with Hoechst 33342 (Beyotime Institute of Biotechnology, Beijing, China) for 5 min in the dark. Following three washes, the cells were observed using fluorescence microscopy.

NB4 cells were treated with 0.3 µmol/l shikonin for 24 h. Treated and control cells were harvested by centrifugation at 1,000 × g for 5 min then washed and re-suspended with cold PBS. Cells were then stained with Annexin V and propidium iodide for 5–15 min at room temperature using the Annexin V/PI Apoptosis Detection kit (KeyGene, Wageningen, The Netherlands). Apoptosis was analyzed on a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The experiment was repeated at least three times.

The effects of shikonin on cell cycle distribution were detected with a cell cycle kit according to the manufacturer's instructions. Cells treated with 0.3 µmol/l shikonin for 24 h and control cells were collected by centrifugation, followed by washing, fixation and propidium iodide staining. The cell cycle distribution was examined by flow cytometry (BD Biosciences).

Cells were lysed with cold radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology) containing protease inhibitor cocktail for 10 min. Cell proteins were collected by centrifuging at 13,000 × g for 30 min and used for immunoblotting analyses. Cell proteins (60 µg) were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk dissolved in TBS with 20% Tween-20, and then incubated with primary antibodies against caspase-3 (no. 9665; 1:1,000), PARP (no. 9532; 1:1,000), c-Myc (no. 5605; 1:1,000), p-ERK1/2 (no. 4370; 1:1,000), ERK1/2 (no. 4695; 1:1,000), p-p38 MAPK (no. 09-272; 1:1,000), p38 MAPK (no. 9218; 1:1,000), p-JNK (no. 4668; 1:1,000), JNK (no. 9252; 1:1,000), β-actin (no. BM0627; 1:4,000) at 4°C overnight. Following three washes, the membranes were incubated with goat anti-rabbit (no. ZB-2301; 1:4,000) or goat anti-mouse (no. ZB-2305; 1:4,000) IgG horseradish peroxidase-linked secondary antibodies, at room temperature for 1 h. The bands were visualized using Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore). Semi-quantification was performed using Cool Imager software (version. 4.0.1; Viagen Biotech Inc., Los Angeles, CA, USA).

All experiments were repeated at least three times. Data are presented as means ± standard deviation. Statistical analysis was performed with SPSS software (version, 17.0; SPSS, Inc., Chicago, IL, USA). One-way analysis of variance and Student's t-test were used for comparisons. P<0.05 was considered to indicate a statistically significant difference.

NB4 cells were treated with increasing concentrations of shikonin for 24 h and viability assessed using the CCK-8 assay. There was no significant difference between the control and shikonin 0.1 µmol/l group in cell viability (Fig. 1A). However, cell viability was reduced significantly by treatment with shikonin concentrations of 0.2 µmol/l or more. The IC₅₀ value was 0.3 µmol/l. NB4 cells were also treated with 0.3 µmol/l shikonin for 0–36 h. The CCK-8 results demonstrated that this treatment reduced cell viability significantly from 12–36 h (Fig. 1B). These results indicated that shikonin inhibited the viability of NB4 cells in a concentration- and time-dependent manner.

The cell nuclei of untreated NB4 cells stained by Hoechst 33342 were round and uniform, while shikonin treatment resulted in chromatin agglutination, karyopyknosis and nuclear fragmentation (Fig. 2).

NB4 cells were treated with 0.3 µmol/l shikonin for 24 h and the cell cycle was detected by flow cytometry. Compared with the control group, shikonin-treated cells were arrested at the G1 phase. The percentage of cells in the G1 phase increased from 37.3 to 51.8% (Fig. 3). This result indicated that shikonin induced cell cycle arrest of NB4 cells.

The effect of 0.3 µmol/l shikonin on NB4 cell apoptosis was detected by flow cytometry at 24 h. The percentage of apoptosis cell was increased significantly by shikonin treatment (Fig. 4).

Western blotting was used to detect the apoptosis-related proteins PARP and caspase-3. The expression of cleaved PARP and caspase-3 was increased following treatment with 0.3 µmol/l shikonin for 24 h, as compared with the control group (Fig. 5), supporting the induction of apoptosis by this treatment.

To investigate possible mechanisms for shikonin-induced apoptosis in NB4 cells, the authors assessed the levels of MAPKs and the expression of c-Myc. Shikonin increased the phosphorylation of p38 MAPK and JNK significantly, and inhibited ERK phosphorylation (Fig. 6). However, the expression of total ERK, p38 MAPK and JNK was not affected by shikonin. Meanwhile, the expression of c-Myc was significantly decreased by treatment with shikonin.