Cucurbitacin B, glutamine, RPMI 1640 medium, fetal calf serum (FCS), penicillin, streptomycin and 0.25% trypsin were purchased from Sigma Aldrich; Merck KGaA (Darmstadt, Germany). Antibodies against ERK1/2 (cat. no. 9102), phospho-ERK1/2 (cat. no. 9101), p38 (rabbit mAb, cat. no. 8690), phospho-p38 (rabbit mAb, cat. no. 4511), JNK (cat. no. 9252), phospho-JNK (mouse mAb, cat. no. 9255), MMP-2 (rabbit mAb, cat. no. 87809), MMP-9 (cat. no. 852) (all from Cell Signaling Technology, Danvers, MA, USA), Bcl-xL (cat. no. sc-8392), Bcl-2-associated death promoter (Bad) (cat. no. sc-8044), Bax (cat. no. sc-4239), Bcl-2 (cat. no. sc-509), VEGF (cat. no. sc-4571), caspase-9 (cat. no. sc-81663), caspase-8 (cat. no. sc-81656), caspase-3 (cat. no. sc-176260), p-JAK2 (cat. no. sc-34479), JAK2 (cat. no. sc-34479), p-STAT3 (cat. no. sc-56747) and STAT3 (cat. no. sc-482) (all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) were used for expression analysis. All other reagents used in the study were purchased from Sigma Aldrich; Merck KGaA, unless otherwise stated.

Human osteosarcoma cell line, U-2 OS was obtained from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology (Chinese Academy of Sciences, Shanghai, China). The cells were cultured and maintained in RPMI-1640 medium supplemented with 10% FCS, 2 mM glutamine, streptomycin (100 µg/ml) and penicillin (100 U/ml) at 37°C in a humidified atmosphere with 5% CO₂.

OS cells were seeded into 96-well plates (5×10⁵ cells/well) and incubated at 37°C for 24 h. On reaching 70% confluence, the cells were treated with different concentrations of cucurbitacin B (20, 40, 80 and 100 µM) for 24 h. Cells were then incubated at 37°C with MTT for 4 h. The formazan crystals formed were dissolved with dimethyl sulfoxide and the absorbance was measured at 570 nm using a Thermo Multiskan Spectrum (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

OS cells were incubated at 37°C with various concentrations of cucurbitacin B (20, 40, 80 and 100 µM) for 24 h. Following incubation, cells were treated with 0.25% trypsin, washed twice with ice-cold PBS and harvested for detection of apoptosis using an Annexin V-FITC apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer's instructions. The cells were washed in PBS at 1×10⁶ cells/ml and suspended at 1×10⁵/100 µl binding buffer (BD Biosciences) containing FITC-conjugated anti-Annexin V antibody (provided in the kit) and PI, and analyzed for fluorescence using a flow cytometer (FACSCalibur; BD Biosciences).

Morphological changes of the U-2 OS cells following treatment with cucurbitacin B was determined by Hoechst staining. Briefly, the OS cells were seeded at a density of 5×10⁴ cells per well in a 6-well plate and cultured at 37°C for 12 h and treated with cucurbitacin B (20, 40, 80 and 100 µM) for 48 h. The cells were then fixed with 4% paraformaldehyde in PBS for 10 min, washed with PBS and stained with Hoechst 33258 (5 mg/l) for 15 min at room temperature in the dark. The cells were observed under a a fluorescence microscope (Nikon Eclipse TiS coupled with NIS-Elements imaging software; Nikon Corporation, Tokyo, Japan). The cells undergoing apoptosis were characterized by condensed or fragmented nuclei.

The influence of cucurbitacin B on the cell migration of U-2 OS cells was assessed. In brief, 5×10⁵ cells were maintained in 10 cm Petri plates and incubated for 24–72 h until they reached complete confluency and were wounded using a 200 µl pipette tip. All cells in the plates were treated with final concentrations of cucurbitacin B (25, 50 and 100 µM; 3 plates for each concentration) and then were incubated at 37°C in fresh culture medium for 24 h. The migration of the cells in the wounded area was measured as previously described (33). Cell migration was determined as a percentage of the cell-free area compared with the area of the initial wounded area. The fields were photographed using an inverted microscope (Nikon Eclipse TS2; Nikon Corporation).

Gel zymography was performed to evaluate the activities of MMP-2 and MMP-9. Sample preparation was done as previously described by Mizoguchi et al (34). Samples were run on SDS-PAGE (10%) gels that contained 0.1% gelatin under non-reducing conditions. Triton X-100 (2.5%) was used to wash the gels to remove SDS, then the gels were washed further in incubation buffer (50 mM Tris HCl, 0.15 M NaCl, 10 mM CaCl₂) (25±2°C) for 30 min and incubated (24 h, 37°C). Following incubation, the gels were stained with Coomassie Brilliant Blue (1% Coomassie Brilliant Blue G-250, 30% methanol and 10% acetic acid) for 3 h and then were destained (7% acetic acid, 40% methanol) until clear bands representing gelatinolysis were seen against dark background. By using an Atto Densitograph Software Library Lane Analyser (Atto Instruments, Inc., Rockville, MD, USA) the total activity was determined.

Following treatment with cucurbitacin B (25, 50 and 100 µM), U-2 OS cells were washed twice with ice-cold PBS and centrifuged at 10,000 × g for 5 min at 4°C. The cells were treated in ice-cold hypotonic lysis buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl₂, 0.2 mM KCl, 0.5 mM dithiothreitol, 0.2 mM phenylmethyl-sulphonylfluoride), vortexed for 2 min, centrifuged at 12,000 × g for 10 min at 4°C and the supernatants were collected and assayed for protein content using the Bradford method (35). Equal amounts of protein samples (60 µg) were subjected to electrophoresis in 12% SDS-PAGE and the bands were then transferred to nitrocellulose membranes (GE Healthcare Lifesciences, Chalfont, UK). The membranes were washed and blocked using blocking buffer (5% non-fat dry milk/0.1% Tween-20 in TBS) for 1 h at room temperature. They were then incubated overnight at 4°C with respective primary antibodies followed by incubation at room temperature for 40 min with horseradish peroxidase-conjugated-secondary antibody (mouse mAb; Cell Signaling Technology, cat. no. 7076; dilution 1:1,000). The immunoreactive bands were detected by enhanced chemiluminescence procedure (GE Healthcare Lifesciences). Protein expression levels were normalized with the expression levels of β-actin using anti-β-actin antibody (AC-15) from Santa Cruz Biotechnology, Inc. (cat. no. sc-69879; dilution 1:1,000). The positive signals from the immunoblots (n=3) were quantified using Image Lab Software (v5.1) (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

The data are presented as mean ± standard deviation, from three or six individual experiments. The values were analyzed by one-way analysis of variance followed by Duncan's Multiple Range Test. All statistical analyses were performed using the SPSS software (version 22.0; IBM SPSS, Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

The viability of osteosarcoma cells following treatment with cucurbitacin B was determined by MTT assay, and the findings are presented in Fig. 1. Osteosarcoma cells treated with different concentrations of cucurbitacin B (20, 40, 80 and 100 µM) exhibited reduced cell proliferation in a concentration-dependent manner. The viability percentage of U-2 OS cells treated with cucurbitacin B at 20 µM was 83%, whereas following the 100 µM dose, the cell viability significantly decreased to 21% (P<0.05; Fig. 1).

The results of MTT assay revealed that cucurbitacin B may effectively reduce the viability of U-2 OS cells. The present study further analyzed whether cucurbitacin B reduced viability by inducing apoptosis. Annexin V/PI staining assay was performed to determine apoptotic cell counts. Exposure to cucurbitacin B was identified to significantly increase the apoptotic cell counts (P<0.05; Fig. 2), in a dose-dependent manner with 100 µM concentration exhibiting maximum effects.

Furthermore, Hoechst 33258 staining was performed to assess morphological changes in the U-2 OS cells following cucurbitacin B treatment. Following treatment for 48 h, the chromatin of U-2 OS cells was condensed and appeared to be brighter and deeper. Notably, treatment with cucurbitacin at higher concentrations of 80 and 100 µM demonstrated deeper staining of nuclear chromatin than the lower doses (20 and 40 µM). Highly condensed and stained chromatin indicates characteristics of apoptotic cells, thus suggesting that cucurbitacin B effectively induced apoptosis in U-2 OS cells. The percentage of apoptotic cells increased significantly following cucurbitacin B treatment (20, 40, 80 or 100 µM) compared with the control (P<0.05; Fig. 3).

Following incubation with cucurbitacin B (25, 50 and 100 µM) for 24 h, the area of the wound was measured. In the cells that were not treated with cucurbitacin B, the distance between the scratches was significantly reduced when compared with U-2 OS cells that were exposed to cucurbitacin, in a dose-dependent manner (P<0.05). In the OS cells that were incubated with cucurbitacin B the cell free area was evidently increased, the distance increased with concentration of cucurbitacin B (Fig. 4). The growth and movement of the cells in the cell free scratch area observed in cells that were not treated with cucurbitacin suggests cell division and migration were occuring, which were supressed following cucurbitacin B treatment.

It has been established that MMPs principally contribute to the invasion and metastasis of tumor cells (36–38). In the present study, to further assess whether cucurbitacin inhibits MMPs, the expression of MMP-2 and −9 were determined by gel zymography and western blot analysis. The result of the current study indicated that cucurbitacin B exposure at all assessed concentrations caused marked inhibitions in the expressions of MMPs (Fig. 5A). The zymography analysis revealed significantly supressed activities of MMP-2 and −9 in a dose-dependent manner (Fig. 5B). Furthermore, western blot analysis revealed a markedly decreased expression level of MMP2 and MMP9 in cucurbitacin B-treated cells. However, relatively little inhibition was observed following treatment with 25 µM cucurbitacin when compared with higher doses, 50 and 100 µM. Furthermore, marked suppression of VEGF levels was also observed following cucurbitacin B exposure in a dose-dependent manner (Fig. 5C). These observations suggest that cucurbitacin B was able to markedly supress cell migration and inhibit angiogenesis, potentially by downregulating the expression of MMP2, MMP9 and VEGF.

Apoptosis is a coordinated network of genes that lead to cell death and is the target pathway in many anticancer therapies. Anticancer drugs are known to induce apoptosis by targeting cells that harbour genetic damage or that divide inappropriately (39). The present study assessed the influence of cucurbitacin B on the expression of apoptotic pathway proteins in U-2 OS cells to investigate the molecular events associated with cucurbitacin-induced cell death. A significant increase in the expression of caspase-3, −8 and −9 in the U-2 OS cells exposed to cucurbitacin B was observed following western blot analysis (P<0.05; Fig. 6). The activation of caspase-9 and −8 have previously been documented, suggesting the involvement of intrinsic and extrinsic apoptotic pathways that subsequently lead to the activation of caspase-3 (40). Therefore, in the present study the enhanced expression of caspases by cucurbitacin B indicates an increased level of apoptosis in U-2 OS cells.

Furthermore, the expression of Bcl-2 family members was also assessed under the influence of cucurbitacin B. The Bcl-2 family are key proteins in the regulation of intrinsic mitochondrial pathways of apoptosis, specifically controlling the release of cytochrome c, which further regulates the caspases cascade (41,42). The Bcl-2 family comprises anti-apoptotic (Bcl-2 and Bcl-xL) and pro-apoptotic proteins [Bax, Bcl-2 antagonist killer (Bak) and Bad] (43). Cucurbitacin B exposure resulted in a multi-fold increase in the Bad and Bax protein expression (P<0.05; Fig. 6B) with significant downregulation of Bcl-2 and Bcl-xL expression (P<0.05; Fig. 6B). The upregulated expression of Bax and Bad indicates the activation of the apoptotic pathway. Therefore, with enhanced levels of pro-apoptotic proteins, cucurbitacin B aids in elevated levels of caspases, which eventually induces apoptosis.

It has been well documented that activation of MAPK pathways serves a critical role in inhibiting apoptosis. In the present study, in order to elucidate the mechanisms underlying cucurbitacin B-induced apoptosis, the expression levels of MAPK cascade proteins, JNK, p-JNK, p38, p-p38, ERK1/2, and p-ERK1/2 were analyzed by western blot analysis. The results demonstrated an enhanced expression of the proteins in the U-2 OS cells that were not exposed to cucurbitacin (Fig. 7). However, U-2 OS cells treated with cucurbitacin B exhibited marked downregulation in the phosphorylated levels of ERK1/2, p38 and JNK. Whereas substantial decreases were observed in the expression levels of p38 and ERK1/2, JNK and p-JNK levels were decreased to a lesser extent. However, marked inhibitions were observed following cucurbitacin B treatment at all the three assessed concentrations, with 100 µM exerting maximum effects. The results indicated that the downregulation of the MAPK signalling pathway may have aided in induction of apoptosis of the U-2 OS cells.

The JAK/STAT3 pathway serves a pivotal role in the transduction of a multitude of signals that are critically involved in the development and homeostasis in mammals (44). STAT3 has been reported to be constitutively activated in different types of cancer in humans (45). It has been demonstrated that the inhibition of STAT3 leads to apoptosis and inhibition of cancer cell proliferation (46). In the present study, western blot analysis was performed to assess the expression level of phosphorylated forms of JAK2 and STAT3 following exposure to cucurbitacin B. The results of the current study indicate that cucurbitacin B treatment significantly reduced the levels of activated JAK2 and STAT3 (P<0.05; Fig. 8). Cucurbitacin B at 100 µM concentration caused a multi-fold decrease in the expression of STAT3 and JAK2. Furthermore, the inhibition of STAT3 and JAK2 expression was dose-dependent. Therefore, these observations indicate that cucurbitacin significantly inhibited the STAT3/JAK2 pathway and this may have a part in the increase rate of apoptosis observed.