All chemicals were purchased from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany) unless otherwise stated. VK4 was purchased from Shanghai Tauto Biotech Co., Ltd. (Shanghai, China), and the chemical structure of VK4 is shown in Fig. 1A. Fetal bovine serum (FBS) was purchased from Hangzhou Sijiqing Biological Engineering Materials Co., Ltd. (Hangzhou, China). The Annexin V-FITC Apoptosis Detection kit, Reactive Oxygen Species assay kit and primary antibodies against Bax, Bcl-2 and caspase-3 were purchased from Beyotime Institute of Biotechnology (Shanghai, China). The β-actin primary antibody and the horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or goat anti-mouse IgG secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

The human osteosarcoma U2OS cell line was obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C and 5% CO₂ in a humidified atmosphere. VK4 was dissolved in dimethyl sulfoxide (DMSO). Cells were treated with 0, 25 or 35 µM VK4 dissolved in DMSO, where the final concentration of DMSO was <1%. DMSO-only treated cells were used as a control.

The effect of VK4 on cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) assay as described previously (9). Briefly, 1×10⁴ U2OS cells/well were seeded in a 96-well plate and treated with 0, 5, 10, 12.5, 20, 25, 35, 40, and 100 µM VK4 for 24 h. Following treatment, MTT reagent (500 µg/ml) was added and cells were incubated at 37°C for a further 4 h. DMSO (150 µl) was subsequently added to dissolve the formazan crystals and the absorbance was read at 570 nm using a microplate reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Data were expressed as the percentage viability, assuming that the absorbance of untreated control cells was 100%. The percentage cell viability was calculated using the following formula: Cell viability (%) = [(A_{570, sample} - A_{570, blank}) / (A_{570, control} - A_{570, blank})] ×100.

U2OS cells were treated with 0, 25 or 35 µM VK4 for 24 h. Shrinkage and detachment of the VK4 treated cells were observed by phase contrast microscopy (Olympus IX71 microscope; Olympus Corporation, Tokyo, Japan) (n=3).

A total of 4×10⁶ U2OS cells were treated with 0, 25 or 35 µM VK4 in the presence or absence of the pan-caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-(O-methyl) -fluoromethylketone (Z-VAD-FMK; 50 µM). Following treatment, cells were harvested, washed with PBS, and resuspended in 200 µl binding buffer containing 5 µl annexin V before they were incubated in the dark for 10 min at room temperature, according to the manufacturer's instructions (Beyotime Institute of Biotechnology). Cells were subsequently labeled with 10 µl PI and the samples were immediately analyzed by the Multicycle AV software using the Beckman Coulter Epics XL Flow Cytometer (Beckman Coulter, Inc., Brea, CA, USA).

Cell cycle analysis was performed as described previously (10). Briefly, 4×10⁶ U2OS cells were treated with 0, 25 or 35 µM VK4 for 24 h. The cells were then washed with PBS and fixed with 70% ice cold ethanol at 4°C overnight. After washing twice with PBS, cells were stained with a PBS solution containing 50 µg/ml of PI and 100 µg/ml RNase A for 30 min in the dark at room temperature. The stained cells were analyzed for cell cycle phase distribution using Beckman Coulter Epics XL (Beckman Coulter, Inc.).

In order to determine the level of ROS generation and the MMP, cells were stained with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and rhodamine 123 (Rho-123), respectively, as described previously (9). Briefly, 4×10⁶ U2OS cells were incubated with 0, 25 or 35 µM VK4 for 24 h. The cells were then harvested, washed with PBS and incubated with DCFH-DA (10 µM) or Rho-123 (5 µg/ml) in the dark for 30 min at room temperature. After washing with PBS, the samples were analyzed for the fluorescence of DCF or Rho-123 by flow cytometry (Beckman Coulter Epics XL; Beckman Coulter, Inc.).

Cell mobility was assessed using an in vitro scratch wound assay. A total of 4×10⁶ U2OS cells were seeded in 6-well plates containing complete medium until 90% confluent. The plate surface was then scraped with a sterile, 200-µl tip and washed twice with PBS to remove remaining cell debris, before the cells were incubated in DMEM medium containing 1% FBS, in the presence or absence of 25 µM VK4 for 24 h. The cells were then observed under an inverted light microscope (Olympus Corporation) and photographed at ×100 magnification. Experiments were performed in triplicate. The distance between the wound edges was measured with a graduated ruler, and the relative scratch breadth of VK4-treated samples was presented as the ratio of the average breadth of treatment cells vs. the average breadth of control cells.

In vitro cancer cell migration activities were evaluated using Transwell microplates. For cell migration, 1×10⁵ cells in 200 µl of DMEM medium containing 1% FBS with or without 25 µM VK4, were seeded in the upper Transwell insert chamber containing a polycarbonate filter (diameter, 6.5 mm; pore size, 8 µm; Costar; Corning Life Sciences, NY, USA). DMEM medium (600 µl) containing 10% FBS (chemoattractant) was added to the lower chamber, and the plates were incubated for 24 h at 37°C in 5% CO₂. Cells that had traversed the membrane were fixed with paraformaldehyde, stained with Coomassie Brilliant Blue R-250 dye (cat. no. 27816; Sigma-Aldrich; Merck Millipore), and were visualized and counted using an inverted light microscope. The cells that did not migrate were removed from the top of the transwell filters by scraping. Experiments were performed in triplicate. The relative migration of cells was presented as the ratio of average cell migration in the control group vs. the average cell migration in the treatment group.

U2OS cells (4×10⁶) were treated with 0, 25 or 35 µM VK4 for 24 h. Adherent and detached cells were collected and total protein was extracted as described previously (10). The protein concentrations were determined using the NanoDrop 1000 Spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc.). Proteins (50 µg) were separated by 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidine difluoride membrane. After blocking with 5% (w/v) non-fat milk for 2 h and washing with tris-buffered saline-Tween 20 solution (TBST), membranes were incubated with Bcl-2 (dilution, 1:1,000; cat. no. AB112), Bax (dilution, 1:300; cat. no. AB026), cleaved caspase-3 (dilution, 1:500; cat. no. AC031) and β-actin (dilution, 1:400; cat. no. sc-134542) primary antibodies overnight at 4°C. After washing with TBST, the blots were incubated with HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG secondary antibodies (dilution, 1:5,000; cat. no. sc-45090) for 1 h at room temperature. Blots were washed with TBST and signals were detected using the ECL plus chemiluminescence kit (Merck Millipore) on X-ray film.

Data are expressed as the mean ± standard error. Differences between two groups were determined using the Student's t-test, while differences among >2 groups were determined using one-way analysis of variance followed by Tukey's multiple comparison test. P<0.05 was considered to indicate a statistically significant difference.

The effect of VK4 on the viability of U2OS cells was evaluated using an MTT assay. As shown in Fig. 1B, VK4 treatment significantly inhibited the growth of U2OS cells in a dose-dependent manner (P<0.05). The IC₅₀ value of VK4 was 25 µM following 24 h treatment. The rate of growth inhibition of U2OS cells was >70% at 35 µM when compared with untreated controls (Fig. 1B). Therefore, concentrations of 25 and 35 µM VK4 were selected for further mechanistic studies.

To examine the effect of VK4 on U2OS cell morphology, cells were treated with increasing concentrations of VK4 for 24 h and cell morphology was observed under a phase contrast microscope. As shown in Fig. 1C, increasing concentrations of VK4 induced severe morphological alterations indicative of cell death, including a reduction in the total number of cells and an increase in the number of detached cells in the culture medium.

Apoptosis and cell cycle arrest are the two major causes of cell growth inhibition (1). The effect of VK4 on U2OS cell apoptosis was determined by annexin V-FITC/PI staining and flow cytometry analysis. As shown in Fig. 2, VK4 induced apoptosis in U2OS cells in a dose-dependent manner. In addition, pretreatment of cells with the pan-caspase inhibitor Z-VAD-FMK, significantly attenuated the apoptotic effects of VK4 (P<0.05). This indicates that VK4 induces apoptosis in U2OS cells via caspase activation.

As shown in Fig. 3, a dose-dependent increase in the percentage of cells arrested in S phase was observed following treatment with VK4. Treatment with 25 and 35 µM VK4 significantly increased the percentage of cells in S phase from 23.73±1.94 to 38.9±1.53 and 51.23±0.93%, respectively. A corresponding decrease in the percentage of cells in G0/G1 phase from 45.48±2.43 to 38.13±1.82 and 28.36±2.91%, and G2/M phase from 28.5±1.21 to 22.16±0.78 and 18.26±1.69% was observed following treatment with 25 and 35 µM VK4, respectively (P<0.05).

Intracellular ROS generation in U2OS cells was evaluated by flow cytometry analysis using DCFH-DA. As shown in Fig. 4, the level of ROS in U2OS cells treated with 25 and 35 µM VK4 was significantly increased when compared with untreated controls (82.6±1.2 and 95.1±1.8 vs. 71.2±0.9% respectively; P<0.05).

Depolarization of the MMP is a characteristic feature of apoptosis (1). Excessive intracellular ROS production has been demonstrated to induce apoptosis by disrupting the MMP (15). In order to investigate the role of ROS in VK4-mediated apoptosis, the MMP in U2OS cells was examined using Rho-123 and flow cytometry analysis. As shown in Fig. 5, a significant reduction in MMP was observed in cells following treatment with 25 and 35 µM VK4 when compared with untreated controls (79.3±1.75% and 53.40±2.9% vs. 93.03±1.35%, respectively; P<0.05).

The effect of VK4 on the migration of U2OS cells in vitro was determined using wound healing and Transwell migration assays, respectively. As shown in Fig. 6A and B, the distance to the center of the ‘wound’ was significantly reduced in cells incubated with 25 µM VK4 for 24 h when compared with untreated controls. As shown in Fig. 6C and D, the migration rate of cells was significantly reduced following treatment with 25 µM VK4 when compared with untreated controls (P<0.05). Taken together, these data indicate that VK4 inhibits the migration of U2OS cells in vitro.

ROS generation and mitochondrial membrane dissipation are characteristic features of mitochondrial apoptosis (11). Therefore, in order to investigate the molecular mechanisms underlying the pro-apoptotic effects of VK4 on U2OS cells, the protein expression levels of Bcl-2, Bax and caspase-3 were examined. As shown in Fig. 7, VK4 significantly increased the protein expression of pro-apoptotic protein Bax, and significantly decreased the expression of anti-apoptotic protein Bcl-2 in a dose-dependent manner when compared with untreated controls (P<0.05). Cleavage of caspase-3 is a major hallmark of apoptosis (1). Therefore, the protein expression of cleaved caspase-3 in VK4-treated U2OS cells was examined. As shown in Fig. 7, VK4 treatment significantly increased the expression of cleaved caspase-3 in a dose-dependent manner, when compared with untreated controls (P<0.05). Taken together, these results indicate that VK4 may induce the intrinsic apoptosis pathway in U2OS cells.