The human ATC cell line 8505C was kindly provided by Dr Jihua Han (The Third Affiliated Hospital of Harbin Medical University, Harbin, China). Cells were cultivated in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in a humidified incubator containing 5% CO₂. SOV was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Antibodies against Ki-67 (catalog no. 9449S) were provided by Cell Signaling Technology, Inc. (Danvers, MA, USA).

Cell viability was assessed with the Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Briefly, cells were cultured in 96-well plates with RPMI 1640 medium overnight prior to treatment with SOV (0, 0.5, 1, 2, 4 and 8 µM) for 1–6 days and incubated at 37°C. The cells were seeded between 500 and 3,000 cells/well, in 100 µl culture medium, depending on the culture times tested (1–6 days). After the appropriate culture time, 10 µl CCK-8 solution was added to each well for 1 h, prior to measuring the absorbance at 450 nm with a microplate reader. The half maximal inhibitory concentration (IC₅₀) values were calculated according to the Reed-Muench method (19). Experiments were performed three times.

Cells were seeded in 6-well plates at a density of 500 cells/well and incubated overnight at 37°C. They were then treated with various concentrations of SOV (0, 0.5, 1, 2, 4 and 8 µM) for 14 days and incubated at 37°C. Each treatment was carried out in triplicate. Plates were incubated for 14 days. Medium containing the appropriate concentration of SOV was replaced twice weekly. Eventually, plates were washed twice with cold PBS, fixed with 4% paraformaldehyde for 10 min, and stained with 3% crystal violet. Colonies containing ≥50 cells were scored.

Cell cycle progression of 8505C cells was analyzed using a Cell Cycle kit (catalog no. 558662; BD Biosciences, San Jose, CA, USA). Briefly, following treatment with various concentrations of SOV (0, 0.5, 1, 2, 4 and 8 µM) at 37°C for 48 h, cells were harvested. A total of 1×10⁶ cells were incubated with reagents A-C, according to the manufacturer's protocol, and cells were subjected to flow cytometry. Experiments were performed three times.

Cell apoptosis was analyzed with the fluorescein isothiocyanate (FITC)/Annexin V Apoptosis Detection kit I (BD Biosciences), according to the manufacturer's protocol. Briefly, cells were treated with various concentrations of SOV (0, 2 and 4 µM) and incubated at 37°C for 48 h. Subsequently, cells were washed twice with cold PBS, resuspended in 1X Binding Buffer at a concentration of 1×10⁶ cells/ml, and 100 µl of each cell solution was transferred to individual 5 ml culture tubes. A volume of 5 µl FITC-conjugated Annexin V and 5 µl propidium iodide were added to the tubes, which were gently vortex-mixed, prior to 15 min incubation in the dark at room temperature. A volume of 400 µl 1X Binding Buffer was then added to each tube. The samples were immediately analyzed by flow cytometry (<1 h). Experiments were performed three times.

Δψm assessment. The lipophilic cationic dye JC-1 was used to measure alterations in Δψm. The JC-1 probe was provided by Beyotime Institute of Biotechnology (Haimen, China) and the assay was performed according to the manufacturer's protocol. Briefly, cells were incubated with SOV (0, 2 and 4 µM) in 6-well plates at 37°C for 48 h, prior to adding 5 µg/ml JC-1 for 20 min at 37°C. After incubation with JC-1, cells were washed twice with PBS. The Δψm was assessed by determining the dual emissions from mitochondrial JC-1 monomers (green, 490 nm stimulated luminescence and 530 nm emission light) and aggregates (red, 525 nm stimulated luminescence and 590 nm emission light) using a confocal laser scanning microscope. An increase in the green/red fluorescence intensity ratio indicated mitochondrial depolarization. The relative Δψm was calculated according to the following formula: Experimental ratio value/Control ratio value ×100. Experiments were performed three times.

Female nude mice (5 weeks old; 16–18 g; Beijing Vital River Laboratories Animal Technology Co., Ltd., Beijing, China) were housed at a constant temperature (23°C) and relative humidity (60%) with a fixed 12-h light-dark cycle, and free access to food and water. After 1 week of feeding in the new environment, mice were then used to generate a subcutaneous tumor model. All surgical procedures and animal care protocols were approved by the Ethics Committee of the First Affiliated Hospital of Harbin Medical University (Harbin, China). The study also complied with institutional guidelines for animal care. An 8505C cell suspension (100 µl) in serum-free RPMI 1640 culture medium (1×10⁶) was subcutaneously injected into the flanks of nude mice. Tumor formation and tumor size were monitored daily. When tumors were ~4 mm in diameter, animals were randomly divided into three groups, according to their similar average tumor sizes (n=4 mice/group). Mice received daily 200 µl intraperitoneal injections of either PBS (control) or SOV (5 or 10 mg/kg) in PBS. The doses and methods were based on our preliminary experiments and previous reports (17,20). Tumor size was assessed twice weekly at the skin surface and calculated as follows: Tumor volume=(width)² × (length/2). After 4 weeks treatment, tumors were harvested, weighed and their volume was measured.

Immuno histochemical analyses of the tumors were performed using an anti-Ki-67 antibody, in order to detect cancer cell proliferation. Briefly, tumor paraffin sections (4 µm) were prepared by fixation with 10% formaldehyde solution for 12 h at room temperature, washing with water for 24 h, dehydration, transparency, embedding with paraffin, sectioning and baking for 30 min at 60°C. Following dewaxing, rehydration and antigen retrieval, the tumor sections were blocked in 3% bovine serum albumin (catalog no. 37520; Thermo Fisher Scientific, Inc.) at room temperature for 2 h, and incubated overnight at 4°C with primary antibody against Ki-67 (1:200; catalog no. 8112S; Cell Signaling Technology, Inc.). Sections were subsequently incubated at room temperature for 30 min with SignalStain^® Boost Detection Reagent (HRP, Mouse) (catalog no. 8125S; Cell Signaling Technology, Inc.), then examined by light microscopy (magnification, ×400).

Cell apoptosis was determined in situ with a terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) cell apoptosis detection kit (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. Briefly, 4 µm tumor sections were dewaxed with xylene twice for 5 min, and soaked in 100% ethanol for 5 min, 90% ethanol for 2 min and 70% ethanol for 2 min. Sections were rinsed with distilled water for 2 min and incubated with 20 µg/ml proteinase K without DNase at 37°C for 15 min. They were eventually washed three times with PBS, and exposed to 50 µl TUNEL working fluid. A fluorescence microscope was used to capture images of 400 high-power fields from the slides. The apoptosis index (%) was calculated according to the following formula: Apoptosis index=Number of apoptotic cells/Total number of nucleated cells ×100. Experiments were performed three times.

GraphPad Prism 7.0 (GraphPad Software, Inc., La Jolla, CA, USA) was used for statistical analysis. Data are expressed as the means ± standard deviation. The differences among samples were evaluated by one-way analysis of variance followed by Dunnett's test. P<0.05 was considered to indicate a statistically significant difference.

The 8505C cell line was cultured with various concentrations of SOV (0.5, 1, 2, 4 and 8 µM) or without SOV (control group) for 1–6 days. The cell survival rate was determined using the CCK-8 kit (Fig. 1A). SOV inhibited the viability of 8505C cells, and exhibited a stronger effect at higher concentrations (Fig. 1B). The IC₅₀ values of SOV for 8505C growth were 3.76, 3.55, 3.23, 1.62, 0.85 and 0.80 µM on days 1–6, respectively (Fig. 1C-I). The mean IC₅₀ was 2.30 µM.

The effects of SOV on the clonogenic survival of 8505C cells were evaluated using colony formation assays. The 8505C cells were exposed to increasing concentrations of SOV (0.5, 1, 2, 4 and 8 µM) or culture medium for 14 days. A decrease in the number of ATC colonies following SOV treatment was observed in a concentration-dependent manner (Fig. 2A and B). Concentrations of SOV ≥1 µM inhibited >50% of 8505C cell colony formation compared with in the control group (P<0.01), and 8 µM SOV inhibited 98% of the colony formation (P<0.001).

In order to explore the anti-proliferative mechanism of SOV, 8505C cell cycle progression was assessed following treatment with SOV. Briefly, 8505C cells were cultured in the presence of 0, 2 or 4 µM SOV, according to the mean IC₅₀ for 48 h. Flow cytometric analysis revealed that SOV blocked the progression of 8505C cells beyond the G₂/M phase (Fig. 3A and B). Treatment with 4 µM SOV resulted in the accumulation of 40% of cells in the G₂/M phase, whereas only 10% of cells in the control group were in the G₂/M phase (P<0.001). These data suggested that SOV may lead to G₂/M phase arrest in 8505C cells.

Following treatment with SOV for 48 h, the apoptotic rate of 8505C cells treated with SOV was significantly higher than that of the control group (P<0.01, Fig. 4A and B). Treatment with 2 µM SOV for 48 h increased the percentage of apoptotic 8505C cells to 20%, and 4 µM SOV resulted in ~40% apoptotic cells. These data suggested that SOV may induce 8505C cell apoptosis in a concentration-dependent manner.

The effects of SOV on Δψm were then assessed. The 8505C cells treated with 2 or 4 µM SOV for 48 h exhibited lower red and higher green fluorescence than those of the control group, and Δψm alterations were observed in a concentration-dependent manner (Fig. 5A). In addition, 60 and 80% of cells exhibited green fluorescence (early apoptotic) in the groups treated with 2 and 4 µM, respectively (Fig. 5B).

Following treatment of nude mice with SOV, or PBS as a control, tumor volume was calculated by assessing tumor diameter weekly. The results demonstrated that the tumor volume in the SOV group was smaller than that in the control group from the first week, and the difference in tumor volume between groups was positively associated with treatment duration. In the two SOV intervention groups, the tumor volume in the high-concentration group (10 mg/kg) was smaller than that in the low-concentration group (5 mg/kg; P<0.05, Fig. 6A and B). This suggested that treatment with SOV significantly inhibited tumor growth compared to that in the control group. Therefore, SOV may inhibit the growth of ectopic ATC.

In order to detect 8505C cell proliferation after SOV or PBS treatment, the expression of the proliferative biomarker protein Ki-67 was detected by immunohistochemistry. The results revealed that the expression of Ki-67 in the tumor tissues of the SOV group was significantly lower than that in the control group. In addition, Ki-67 expression in the high-SOV group (10 mg/kg) was significantly lower than that in the low-SOV group (5 mg/kg; P<0.05, Fig. 6C and D). In order to assess the apoptosis of 8505C cells, an in situ TUNEL assay was performed. The results demonstrated that the number of apoptotic cells treated with SOV was higher than that in the control group, and was positively associated with SOV concentration (Fig. 6E and F). These results revealed that SOV inhibited the proliferation and promoted the apoptosis of 8505C cells in vivo.