The MG-63 human osteosarcoma cell line was obtained from Nanjing KeyGen Biotechnology, Co., Ltd. (Nanjing, China). The human umbilical vein endothelial cells (HUVECs) were obtained from the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). The two cell lines were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) (Gibco-BRL, Gaithersburg, MD, USA) at 37°C in an incubator with 5% CO₂. Cannabinoid WIN-55,212-2 was purchased from Sigma-Aldrich (St. Louis, MO, USA) and ADM was purchased from Pharmacia (Milan, Italy).

The cell proliferation assay was performed using a Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) according to the manufacturer's instructions. Briefly, exponentially growing MG-63 cells (6×10³ cells/well, 100 µl) were seeded into 96-well plates and cultured overnight using the conditions described above for the cell culture. Five experimental groups were established: ‘Cannabinoid,’ ‘ADM,’ ‘Cannabinoid + ADM,’ ‘Control’ and ‘Blank.’ The adherent cells in ‘Cannabinoid’ and ‘ADM’ were exposed to 100 µl per well of 20 µM cannabinoid WIN-55,212-2 and 20 µM ADM, respectively, dissolved in RPMI-1640 fresh medium (Gibco-BRL). Cells in ‘Cannabinoid + ADM’ were exposed to a mixture of the above cannabinoid and ADM solutions at a ratio of 1:1 of cannabinoid:ADM (100 µl/well). Fresh medium without drugs (100 µl/well) were added into ‘Control’ as the control group. The wells for ‘Blank’ which contained medium without either drugs or cells (100 µl/well) served as the zero adjustment. The plates were then incubated for 23 h. CCK-8 (10 µl) was subsequently added to each well and the cells were further incubated at 37°C for 1 h. Absorbance at 450 nm was measured using a microplate reader (Infinite® M200 PRO, Tecan GmbH, Grödig, Austria). The viable rate of cells in each group was calculated using the following equation:

In which optical density (OD) (control) was obtained from ‘Control,’ OD (treated) was obtained from the groups with cells in the presence of drugs, and OD (adjustment) was obtained from ‘Blank.’ The assay was performed in triplicate.

The cell migration was assessed by scratch assay. MG-63 cells were seeded at a density of 5×10⁵ cells per well in a 6-well plate and grown overnight to confluence on the plates. A single scrape was made in the confluent monolayer using a sterile pipette tip. The monolayer was washed with PBS [Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China] and 2 ml serum-free medium containing 20 µM cannabinoid WIN-55,212-2, 20 µM ADM or ‘Cannabinoid + ADM’ (Gibco-BRL) was added. Fresh medium without any drugs was used as the control. Serial photographic images of the same scraped section were captured at 0, 12, 24 and 36 h using a Leica TCS 4D microscope (Leica Camera AG, Wetzlar, Germany).

The invasive capacity of MG-63 cells was measured using a Transwell® chamber (Corning, NY, USA). All reagents and the Transwell® chamber were pre-incubated at 37°C. The suspension of exponentially growing MG-63 cells (2×10⁵ cells/ml) was placed into the upper compartment in serum-free medium. Subsequently, 20 µM cannabinoid WIN-55,212-2, 20 µM ADM or ‘Cannabinoid + ADM’ were separately added into the upper compartments. The complete medium containing 10% FBS was placed into the lower compartment and served as chemoattractant. After 48 h of incubation at 37°C with 5% CO₂, non-invading cells were removed using cotton swabs. Invading cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 40 min, stained with 0.1% crystal violet (Sigma-Aldrich) for 30 min and counted in six random image fields by microscopy (TCS 4D; Leica Camera AG). The invasion rates were calculated as follows:

In which N (control) was the number of invading cells in the control group and N (treated) was the number of invading cells in the groups with cannabinoid and/or ADM treatment.

The angiogenic properties of MG-63 cell-conditioned medium were assessed using the angiogenesis assay with HUVECs in vitro. Exponentially growing MG-63 cells (1×10⁵ cells/ml) were seeded in a 24-well plate and incubated with 20 µM cannabinoid WIN-55,212-2, 20 µM ADM or ‘Cannabinoid + ADM’ for 24 h at 37°C with 5% CO₂. Conditioned medium was obtained by collecting the supernatant following centrifugation at 1,000 × g for 10 min. Exponentially growing HUVECs were cultured in the mixed medium containing equal amounts of conditioned and serum-free medium, and then seeded into 12-well plates precoated with Matrigel™ at a density of 5×10⁴ cells/ml. Plates were incubated at 37°C with 5% CO₂ for 8 h. The tube formation of HUVECs was observed by microscopy (TCS 4D; Leica Camera AG) and counted in five random image fields. The assay was performed in triplicate.

Following treatment with 20 µM cannabinoid (WIN-55,212-2), 20 µM ADM or ‘Cannabinoid + ADM’ and incubation for 24 h at 37°C with 5% CO₂, MG-63 cells were harvested. MG-63 cells without treatment served as the control group. The total RNA was extracted from each sample of 1×10⁶ cells using TRIzol® (Invitrogen Life Technologies, Carlsbad, CA, USA) following the manufacturer's instructions. M-MLV reverse transcriptase (Takara Bio Inc., Otsu, Japan) and oligo(dT) were used to synthesize the first-strand cDNA from the total RNA. The mixture was then incubated at 37°C for 2 h, terminated by heating at 95°C for 5 min and stored at −20°C. The transcript levels of Notch-1, matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) were detected by RT-qPCR using a Light Cycler 2.0 (Roche Diagnostics Deutschland GmbH, Mannheim, Germany) using SYBR® Green Master Mix (Toyobo, Osaka, Japan). The RT-PCR program was as follows: 95°C for 10 sec, followed by 40 cycles of 95°C for 10 sec, 56–65°C for 5 sec and 72°C for 10 sec. The GAPDH gene, which served as the reference gene for the internal standard, was amplified with forward and reverse primers. The PCR primers used to detect each gene were synthesized by SBS Genetech Co., Ltd. (Beijing, China) and the sequences and information are listed in Table I. The dissociation curve analysis of amplification products was performed at the end of each PCR reaction to confirm the specificity of the amplification. The data were then analyzed by the 2^−∆∆Ct method (23) to obtain the relative gene expression levels compared with that of the controls.

The expression levels of Notch-1, MMP-2 and VEGF proteins in MG-63 cells were detected by western blot analysis, and β-actin was used as the loading control. As described for the total RNA extraction, MG-63 cells with or without drug treatments were lysed in ice-cold lysing buffer consisting of 50 mM Trizma base (pH 7.4; Sigma-Aldrich), 1% Triton X-100, 150 mM NaCl, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 2 mM sodium pyrophosphate, 25 mM β-glycerophosphate, 1 mM EDTA, 0.5 µg/ml leupeptin and 1 mM sodium vanadate. All reagents were purchased from Sigma-Aldrich. Cell lysates were centrifuged at 13,000 × g for 5 min at 4°C. The supernatant was collected and the protein expressions were measured as follows. The whole cell extracts (50 µg/lane) were separated on 12% SDS-PAGE gels and then transferred to polyvinylidene difluoride membranes. Subsequent to blocking in Tris-buffered saline with 5% (w/v) non-fat dried milk, the membranes were probed with a primary mouse monoclonal anti-human antibodies for Notch-1, MMP-2 or VEGF (Abcam, Cambridge, UK) diluted in blocking buffer to a concentration of 1:1,000 at 4°C overnight. Subsequent to washing three times with Tris-buffered saline with Tween (TBST), membranes were incubated with diluted secondary HRP-labeled goat anti-mouse IgG antibody (Abcam), at a concentration of 1:5,000, conjugated with horseradish peroxidase and detected by enhanced chemiluminescence reagent [Sangon Biotech (Shanghai) Co., Ltd.]. Photographic images of the bands were taken and analyzed by National Institutes of Health Image software (Bethesda, MD, USA).

Statistical analysis was performed with a Student's t-test using SPSS 20.0 (IBM SPSS, Armonk, NY, USA). All data are presented in the form of the mean ± standard deviation. Qualitative data were analyzed using the χ² test. Differences between the treatment and control groups were considered significant at P<0.05.

Cannabinoid and ADM were tested individually and as a combined treatment to ascertain whether these agents inhibited the proliferation of MG-63 cells. The groups, ‘Cannabinoid,’ ‘ADM,’ and ‘Cannabinoid + ADM,’ in which cells were incubated with 20 µM cannabinoid (WIN-55,212-2), 20µM ADM and or the two agents for 24 h, were shown to effectively inhibit the growth of MG-63 cells. The viability rates of cells in the ‘Cannabinoid,’ ‘ADM’ and ‘Cannabinoid + ADM’ treatment groups were 70.86±7.55, 62.87±5.98 and 32.12±3.13%, respectively, which were all significantly reduced (P<0.05) compared with those in ‘Control’ (100%). Notably, the viability rate of cells in the ‘Cannabinoid + ADM’ group was significantly lower (P<0.05) than those in ‘Cannabinoid’ and ‘ADM’ groups (Fig. 1).

MG-63 cells were treated with 20 µM cannabinoid (WIN-55,212-2) and ADM alone or combined, and cell migration was measured by scratch assay. The results demonstrated that the individual and combined treatments of cannabinoid and ADM had time-dependent inhibition effects on the migration of cells. This inhibition was enhanced as time progressed. Compared with the control treatment, the number of cells that had migrated after 24 h to heal the wound was fewer in the cells with cannabinoid and/or ADM treatment, and the scraped sections in these treatment groups were also wider (Fig. 2). Notably, these phenomena were observed most markedly in ‘Cannabinoid + ADM’. In the cell invasion assay, as shown in Fig. 3A, the number of invading cells in the treatment groups with cannabinoid and ADM alone or combined was reduced compared with the control group, and the number of invading cells in ‘Cannabinoid + ADM’ was the lowest in all the three treatment groups. Consistently, the invasion rate of cells in ‘Cannabinoid + ADM’ was significantly lower than those in the groups with the individual treatments of either cannabinoid or ADM (Fig. 3B).

The angiogenic properties of MG-63 cell-conditioned medium were assessed using the angiogenesis assay with HUVECs in vitro. The results showed that tube formation of HUVECs was observed with the drug-free MG-63 cell-conditioned medium. However, tube formation was significantly reduced when the HUVECs were cultured with the cannabinoid and ADM alone, or the combined conditioned medium. The combined treatment reduced the angiogenesis to the greatest extent (Fig. 4).

The expression levels of Notch-1, MMP-2 and VEGF genes were measured by RT-qPCR (Fig. 5). The results showed that the mRNA expression levels of these genes in MG-63 cells with cannabinoid and/or ADM treatment reduced significantly (P<0.05) compared with those in the control group without any drug treatment. Furthermore, the expression levels of Notch-1, MMP-2 and VEGF in cells with the combined treatment of cannabinoid and ADM were significantly lower (P<0.05) compared with cells that received individual treatment of either cannabinoid or ADM. Western blotting further confirmed these downregulation effects (Fig. 6).