Shikonin was obtained from the Shanghai Jinsui Biotechnology Co., Ltd. (Shanghai, China). After being dissolved in dimethyl sulfoxide (DMSO) at a stock solution (0.05 mol/l), shikonin was stored at −20°C. Subsequent dilutions were conducted with culture medium. An equal proportion of vehicle was added to the control cells.

3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) and Hoechst 33342 were obtained from Sigma Chemical Company (St. Louis, MO, USA). An Annexin V/propidium iodide (PI) apoptosis kit was obtained from Invitrogen (Carlsbad, CA, USA). Primary antibodies against Bcl-2, Bax, cleaved caspase-9, cleaved caspase-3, cleaved PARP, cyclin D1, CDK4, JNK, phosphorylated JNK (p-JNK), GAPDH and secondary antibodies (goat anti-rabbit) were obtained from Cell Signaling Technology (Danvers, MA, USA).

NOZ and EHGB-1 (human GBC) cell lines were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences and grown in high-glucose Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY, USA). The media contained 100 U/ml penicillin (HyClone, Logan, UT, USA), and 10% fetal bovine serum (FBS; Gibco) and 100 µg/ml streptomycin. The cells were grown at 37°C in an atmosphere with 5% CO₂.

Cell viability was detected by the MTT assay. NOZ and EHGB-1 cells (2×10³/well) were seeded into 96-well plates and incubated overnight. Then, the cells were treated with shikonin at various concentrations of 0, 0.5, 1, 2, 3 and 4 µmol/l for 24, 48 and 72 h. After treatment, 10 µl of MTT solution (5 mg/ml) was added to each well and incubation was carried out at 37°C for 4 h. The culture medium was then replaced with 100 µl of DMSO. Absorbance of the solution at 490 nm was measured with a microplate reader (BioTek, Winooski, VT, USA). The results represent the average of three parallel samples.

NOZ and EHGB-1 cells were liquated as single cell suspensions and 600 cells were seeded into each well of 6-well plates. Cells were treated with shikonin (0, 0.1, 0.2 and 0.3 µmol/l for NOZ and EHGB-1), and cultured for ~14 days. The cells were then fixed with 4% paraformaldehyde for 15 min, and stained with 0.1% crystal violet (Sigma-Aldrich, St. Louis, MO, USA) for 30 min. The total number of colonies (>50 cells/colony) was manually counted.

Cells were seeded in 6-well plates and treated with shikonin (0, 1, 2 and 3 µmol/l) for 48 h. After adherence, cells were harvested and washed twice with cold phosphate-buffered saline (PBS), then resuspended at a density of 1×10⁶ cells/ml. Next, 300 µl of binding buffer containing 5 µl of Annexin V-FITC and 5 µl of PI (100 µg/ml) was added to the cells, followed by incubation in the dark for 30 min. The samples were determined using flow cytometry (BD Biosciences, San Diego, CA, USA).

NOZ and EHGB-1 cells were treated with shikonin in different concentrations (0, 1, 2 and 3 µmol/l) for 48 h. Cells were harvested, washed with cold PBS, and fixed in 70% ethanol overnight. Then, the cells were washed, added with RNase and PI (Sigma-Aldrich), and incubated in the dark for 30 min. The cells were detected by flow cytometry (BD Biosciences). The percentages of cells in each phase of the cell cycle were analyzed by CellQuest acquisition software (BD Biosciences).

After treatment with shikonin (0, 1, 2 and 3 µmol/l) for 48 h, the NOZ and EHGB-1 cells were washed in cold PBS and added with methanol:acetic acid (3:1) for 10 min. The cells were stained with Hoechst 33342 5 µg/ml for 10 min at 37°C and subsequently observed with a fluorescence microscope (Leica Biosystems, Wetzlar, Germany).

Cells were treated with shikonin (0, 1, 2 and 3 µmol/l) for 48 h, and then harvested, washed and lysed in RIPA buffer (Beyotime Institute of Biotechnology, Beijing, China) and protease inhibitor (Roche Applied Science, Indianapolis, IN, USA) at 4°C for 5 min. The lysates were centrifugated at 14,000 × g for 5 min, the supernatant was collected, and the protein concentration was detected by a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology). The proteins were loaded on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% skim milk, and incubated with primary antibodies against Bcl-2, Bax, cleaved caspase-3, cleaved caspase-9, cleaved PARP, cyclin D1, CDK4 and GAPDH overnight. The membrane was washed with Tris-buffered saline with Tween-20 (TBST), and then incubated with HRP-conjugated goat anti-rabbit secondary antibodies (Abcam, Cambridge, UK) for 1.5 h. The bands were detected with Gel Doc 2000 (Bio-Rad, Hercules, CA, USA).

Four-week-old male athymic nude mice were purchased from the Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). All procedures were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University. NOZ cells were resuspended in PBS (1×10⁶ cells in 0.2 ml), and injected into the right flank of each mouse. Twenty-four hours after inoculation, the mice were randomly divided into three groups (5 mice/group). The control group was injected with vehicle (10% DMSO and 90% PBS) and the others were injected with shikonin (1 or 3 mg/kg) every 2 days for up to 14 days. The body weight was measured every 2 days. On day 15, the tumor tissues were removed and weighed after sacrifice of the mice injected with lethal dose of pentobarbital. The tumor volume (V) = 1/2 × length × width².

All assays were performed three dependent times, and data are expressed as means ± SD. The Student's t-test in GraphPad Prism was applied to assess the difference between two groups (GraphPad Software, San Diego, CA, USA). P<0.05 (*P<0.05, **P<0.01, ***P<0.001) was considered to indicate a statistically significant result.

The cell proliferation was assessed by MTT assay. We discovered an obvious reduction in the viability of shikonin-treated cells (Fig. 2A). The inhibitory concentration (IC)50 for NOZ and EHGB-1 cells was ~2 µmol/l at 48 h treat-ment. The ability of NOZ and EHGB-1 cells to form colonies when treated with shikonin was demonstrated by the colony formation assay (Fig. 2B). The result suggested that shikonin exerted a negative influence on colony formation ability. Furthermore, we found less amount of clone formations in shikonin-treated groups than those in the control groups (Fig. 2C). These results indicated that shikonin obviously suppressed the proliferation and colony forming of NOZ and EHGB-1 cells.

The impacts of shikonin on apoptosis in the NOZ and EHGB-1 cells were detected using Annexin V/PI staining and flow cytometry. As shown in Fig. 3A, the percentage of surviving cells was reduced, whereas the percentage of apoptotic cells was obviously increased (Fig. 3B).

To further confirm the cell apoptosis, we applied Hoechst 33342 staining to examine the change in nuclear morphology. The untreated cells were round with uniformity in chromatin distribution, while vivid chromatin condensation and lobulated nuclear fragmentation were revealed in cells treated with shikonin (Fig. 3C). Furthermore, the proportion of apoptotic nuclei that was obviously increased agreed with the gradually increased shikonin concentration.

Proteins of the Bcl-2 and caspase family play major roles in initiation and maintenance of mitochondrial apoptosis (19). To further explore the molecular mechanism underlying shikonin-mediated apoptosis, we evaluated the quantitative levels of apoptosis-related proteins via western blot analysis, including Bax, bcl-2, cleaved caspase-9, cleaved caspase-3 and cleaved PARP. As shown in Fig. 4A, we observed increased expression of Bax, cleaved caspase-9, cleaved caspase-3 and cleaved PARP and downregulation of Bcl-2. Moreover, the Bcl-2/Bax ratio represents apoptotic activity (20), as cell apoptosis is promoted when the ratio decreases. In the present study, we found a significant decrease in the ratio of Bcl-2/Bax in the shikonin-treated cells (Fig. 4B). We also observed upregulation of p-JNK while no significant change in the expression of JNK was noted.

Together, these results revealed that shikonin may initiate mitochondrial-dependent apoptosis via the JNK signaling pathway in GBC cells.

The percentage of cells in each phase of the cell cycle was determined via flow cytometry. We found an obvious increase in the proportion of G0/G1 cells (Fig. 5B), indicating that shikonin suppressed cell cycle progression in the NOZ and EHGB-1 cells (Fig. 5A). We detected the expression of cell cycle-related proteins cyclin D1 and CDK4, and observed an obvious decrease in the NOZ and EHGB-1 cells (Fig. 5C). Therefore, shikonin may inhibit GBC cell proliferation by triggering G0/G1 phase arrest.

To further detect whether shikonin suppresses tumor growth in vivo, nude mice with palpable tumor xenografts were injected with vehicle (10% DMSO and 90% PBS) or shikonin (1 and 3 mg/kg) every other day. This dose was determined to be effective (21,22) and a non-toxic level; LD50 of shikonin in the mouse is 20 mg/kg when injected in an intraperitoneal manner (23). After injection, the bile and liver were previously found to contain the highest levels of shikonin, and most of the excreted metabolite was transformed (24). As shown in Fig. 6A-D, the shikonin group produced the smaller tumors when compared with the tumor volume of the vehicle group. The results demonstrated that tumor growth was obviously suppressed in the shikonin-treated mice in a dose-dependent manner. Moreover, we found no significant difference in regards to body weight between the vehicle and shikonin-treated groups (Fig. 6E), suggesting that shikonin had no side-effects in nude mice. As shown in Fig. 6F, we detected the upregulation of Bax, cleaved caspase-9, cleaved caspase-3 and cleaved PARP and downregulation of Bcl-2 in the tumor tissues by western blot assay.