U118 and U138 human glioma cell lines were purchased from the Health Science Research Resources Bank (Osaka, Japan). GC, all-trans RA, dimethylformamide (DMF), dimethylsulfoxide (DMSO), dialysis membranes (molecular weight cut-off, 12,000 g/mol) and propidium iodide (PI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fluorescein isothiocyanate (FITC)-Annexin V was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The present study was approved by the Institutional Review Board and Ethics Committee of the Nanjing University (Jiangsu, China).

Preparation of the RA-incorporated GC nanoparticles was performed, as previously described by Jeong et al (10). A solution containing 5 mg RA in 1 ml DMF was slowly added to an aqueous solution containing 40 mg GC in 10 ml deionized water, whilst stirring. The stirring was continued for 20 min under darkened conditions. A dialysis membrane (molecular weight cut-off, 12,000 g/mol) was used to prepare the dialyzed solution in deionized water using a dialysis method for 1 day. The resulting dialyzed solution was lyophilized and analyzed. From the 20 ml solution, prepared by adding deionized water to the dialyzed solution, 100 µl was diluted with 9.9 ml DMSO. A UV-1200 spectrophotometer (Shimadzu Corporation, Kyoto, Japan) was then used to measure the drug contents at 365 nm, and empty GC vehicles were used as blank tests. The drug contents and loading efficiency were calculated using the following equations: Quantity of RA in the nanoparticles / weight of nanoparticles) × 100 and (residual quantity of RA in the nanoparticles / feeding quantity of RA) × 100, respectively.

A Hitachi-S4700 Field Emission Scanning Electron Microscope (Hitachi, Ltd., Toyko, Japan) was used to observe the morphology of the RA-incorporated GC nanoparticles. The RA-incorporated GC nanoparticle solution (one drop) was placed onto a cover glass and dried at room temperature. The gold-coated nanoparticles were observed at 20 kV. A Nano-ZS system (Malvern Instruments, Ltd., Malvern, UK) was used to analyze particle size.

The U118 and U138 human glioma cells were maintained in RPMI 1640 medium (RPMI:ECM, 4:1; Gibco Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) at 37°C in 5% CO₂ in a humidified atmosphere.

Aliquots containing 3×10⁵ cells were seeded into each well of a 96-well plate. The cells were incubated overnight in a 5% CO₂ incubator at 37°C. Following incubation, the RA-incorporated GC nanoparticle solution in DMSO, diluted with RPMI 1640 and supplemented with 10% FBS, was added to each well. Following dilution, this solution was used to treat the tumor cells. RPMI 1640 supplemented with 10% FBS, with 0.1% (v/v) DMSO used as a control. Following 48 h incubation at 37°C, 25 µl MTT [3 mg/ml in phophate-buffered saline (PBS); Sigma-Aldrich] was added to each well, and incubated for a further 4 h at 37°C. To each well 100 µl SDS-HCl solution (10% SDS w/v, 0.01 M HCl; Sigma-Aldrich) was then added and incubated for 12 h at 37°C. An Infinite M200 Pro Reader (Tecan Austria GmbH, Salzburg, Austria) was used to measure the absorbance at 570 nm. The viable cells were expressed as a percentage of the control, and all experiments were performed in triplicate. The following equation was used to calculate the cell viability index: Experimental OD / control OD.

The transfected U118 and U138 cells (2×10⁵ cells per well) from each group were washed twice with PBS. A total of 2 ml lysis buffer, containing 50 mM Tris-HCl (pH 7.4), 137 mM NaCl, 10% glycerol, 100 mM sodium vanadate, 1 mM phenylmethanesulfonyl fluoride, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 1% NP-40 and 5 mM cocktail, was added to the cells (Beyotime Institute of Biotechnology, Haimen, China). A bicinchoninic acid assay method was used to determine the protein concentration. Equal quantities of protein (2 µg) were loaded and separated on an 10% polyacrylamide gel (Merck KGaA, Darmstadt, Germany). A semi-dry method was used to transfer the proteins onto a polyvinylidene difluoride membrane (EMD Millipore, Temecula, CA, USA), which was then blocked with 5% non-fat dry milk overnight at 37°C. Following washing with Tris-buffered saline with Tween 20 (TBST; PerkinElmer, Inc., Waltham, MA, USA), the membrane was incubated for 2 h at 37°C with the following primary antibodies (dilution, 1:1,000): Polyclonal rabbit IgG antibodies against Bax (cat. no. 5023; Cell Signaling, Inc., Danvers, MA, USA), Bcl-2 (cat. no. 2870; Cell Signaling, Inc.), cytochrome c (cat. no. 4280; Cell Signaling, Inc.) and rabbit β-actin (cat. no. ABIN1742508; Wuhan Boster Biological Technology, Ltd., Wuhan, China). The membrane was then washed again with TBST prior to incubation with secondary antibodies for 2 h (polyclonal goat anti-rabbit; cat. no. sc-2034; Santa Cruz Biotechnology, Inc.; dilution, 1:5,000). X-ray autoradiography was performed and the gray scale images (NanoZoomer 2.0-HT slide scanner; Hamamatsu Photonics, Hamamatsu, Japan) were analyzed.

Identification of apoptosis and necrosis in the U118 and U138 cells (2×10⁵ cells per well) was performed with PI and FITC-Annexin V staining, respectively. Treatment of the cells with 10 or 20 µM of RA-incorporated GC nanoparticles for 24 h was followed by washing with PBS. The cell contents were centrifuged at 12,000 × g for 45 min. Following suspension in binding buffer containing 10 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM KCl, 1 mM MgCl₂ and 1.8 mM CaCl₂, with FITC-Annexin V (1 µg/ml), the pellets were incubated for 20 min. PI (10 µg/ml) was then added to stain the necrotic cells under dark conditions and incubation was continued for 10 min. A FACScan flow cytometer (BD Biosciences, San Jose, CA, USA) was used to analyze the cells.

In a 96-well plate, the cells were seeded at a density of 8×10³ cells/well and incubated with the RA-incorporated GC nanoparticles. Following centrifugation for 5 min at 200 × g, the cells were fixed with 80% methanol (Sigma-Aldrich) in PBS for 30 min at room temperature. The plates were dried, and incubated in formaldehyde (Sigma-Aldrich) for 10 min at room temperature, followed by 10 min at 75°C and then at 4°C for 5 min. The cells were then incubated with 3% non-fat dry milk for 1 h at 37°C, followed by incubation with an antibody mixture containing a primary monoclonal antibody targeting ssDNA (F7-26 mouse IgG; cat no. BML-SA264; Enzo Biochem, New York, NY, USA; dilution, 1:1,000) and a horseradish peroxidase-conjugated secondary antibody (polyclonal anti-mouse IgG in goat; cat no. A2304; Enzo Biochem, New York, NY, USA; dilution, 1:5,000) for 30 min. The addition of 2-2′-azino-bis(3-ethylbenziazoline-6-sul-fonic acid) solution permitted the reading of the plates at 405 nm using a standard microtiter reader. The ssDNA was used as a positive control and necrotic cells obtained by hyperthermia were used as a negative control (the cells were heated at 56°C for 1 h and then incubated for 1 h at 37°C).

The cells seeded into 6-well plastic plates (2×10⁶ cells per well) were allowed to reach 70% confluence. The cells were then washed with serum-free medium three times. JC-1 Mitochondrial Membrane Potential Detection kit (Biotium Inc., Hayward, CA, USA) was used to monitor the changes in mitochondrial membrane potential according to the manufacturer's instruction.

The total RNA from U118 and U138 cells was isolated using an RNA isolation kit [Tiangen Biotech (Beijing) Co., Ltd., Beijing, China] according to the manufacturer's instructions. Equal quantities of RNA (300 ng) were used to reverse transcribe the cDNA using an RT kit (Fermentas, Burlington, ON, Canada). Amplification of the cDNA was performed using a SYBR Green Realtime PCR Master Mix kit (Roche Diagnostics, Mannheim, Germany) on a real time fluorescence quantitative instrument (RealPlex 4; Eppendorf Corporation, New York, NY, USA). All primers used were obtained from Parkson Co. (Shanghai, China). For the confirmation of primer specificity, a dissociation curve for each primer pair was analyzed. Each sample was repeated three times for each gene.

The results are expressed as the mean ± standard error of the mean, calculated from the specified number of determinations. SPSS software (version 16.0; SPSS, Inc., Chicago, IL, USA) was used for the statistical analysis of the data. Student's t-test was used to compare individual treatments with their respective control value. P<0.05 was considered to indicate a statistically significant difference.

The exposure of the U118 and U138 human glioma cell lines to RA for 24 h resulted in a dose-dependent inhibition of the cells, as determined using an MTT assay. The experimental concentrations ranged between 5 and 50 µM. The significant concentration for the two cell lines was confirmed as 10 µM, which induced a reduction in OD values of 16±0.6 and 13±0.8% for the U118 and U138 cells, respectively, compared with the control group. Further inhibition in the MTT assay was observed at 20 µM, resulting in 23±2 and 36±3.2% reductions for the U118 and U138 cells, respectively; 30 µM resulting in 63±3.5 and 64±3.43% reductions for the U118 and U138, cells, respectively; and 40 µM, resulting in reductions of 90±10 and 89±10.34% for the U118 and U138 cells, respectively. The half maximal inhibitory concentration (IC₅₀) values were calculated as 24.2±2.8 µM for the U118 cells and 23.9.1±2.16 µM for the U138 cells.

To determine the effect of a single administration of RA, a series of experiments were performed using the mean IC₅₀ concentration of 25 µM, following cell growth period of 4 days. Daily MTT assays were performed over the 4 days, which indicated that growth inhibition for the two cell lines (Fig. 2A and B) was present during this period, with a maximum effect observed on day 4.

The above results correlated with those of the trypan blue exclusion assay. A decrease in cell number was observed following exposure to RA in a time-dependent manner (Fig. 2C and D), which was assessed by cell counting. These results suggested that RA exerted its inhibitory effects on the proliferation of glioma cell lines in a dose- and time-dependent manner.

RA-incorporated GC nanoparticles and empty GC vesicles were transfected into U118 and U138 human glioma cells, and changes in the mRNA and protein expression levels of Ezh2 following transfection were analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR; Fig. 3A and B) and western blotting (Fig. 3C), respectively. At 24 h post-transfection with the RA-incorporated GC nanoparticles (25 µM), the expression levels of Ezh2 were significantly reduced in the two cell lines, compared with the control group. By contrast, the protein expression levels in the glioma cells were markedly higher, compared with those in the control group. The silencing of Ezh2 by RA lasted for >72 h following RA-incorporated GC nanoparticle transfection (Fig. 3B). These results suggested that, following transfection of RA at 25 µM for 24 h, the expression levels of Ezh2 are effectively inhibited.

In the present study, apoptotic cell death led to a reduction in glioma cell growth, confirmed using flow cytometric analysis and an ssDNA detection assay. Apoptosis was determined on the appearance of a hypodiploid DNA peak following PI staining of the nuclei of the total cell population (adhering + detached cells), determined using flow cytometric analysis. In the U118 control culture, which was grown in a medium containing empty GC vesicles, only 2.05±1.01% of the cells underwent spontaneous apoptosis (Fig. 4A), following 24 h of incubation. The effective concentration of RA (10 µM) in the MTT assay caused an induction of apoptosis at higher levels than that observed in the control cell culture (5.89±3.98%; Fig. 4B). The 24 h cell cultures, which were exposed to the IC₅₀ concentration of RA caused an induction of apoptosis in 59.54±5.42% of the total cell population (Fig. 4C). Similar results were observed in the U138 cells, in which the percentage of apoptotic cells in the control group was 1.79±0.23%, and the percentage following exposure to RA was increased to 9.86±8.89% at a concentration of 10 µM, and 47.54±14.5 at a concentration of 25 µM RA (data not shown).

JC-1 staining was used to detect changes in mitochondrial membrane potential in the U118 and U138 human glioma cell lines. As shown in Fig. 5A, an increase in the concentration of RA in the RA-incorporated GC nanoparticles between 10 and 25 µM significantly reduced the mitochondrial membrane potential in the U118 cells. Therefore, the results suggested that treatment with RA lead to inhibition of the expression of Ezh2, which in turn affected the mitochondrial membrane potential. The present study also demonstrated, following western blot analysis (Fig. 5B), that translocation of B cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) between the mitochondria and the cell cytosol occurred. The presence of Bax and Bcl-2 also caused the release of cytochrome c in the mitochondria, the results of which are concordant with those of Zhang et al (25). Cytochrome c is important in the induction of apoptosis. Similar results were obtained in the U138 human glioma cell lines.

To further investigate the inhibition of proliferation caused by RA-incorporated GC nanoparticle treatment, the U118 and U138 cells were individually transfected with 10 or 25 µM RA, or empty GC vesicles as a control. The cell cycle was analyzed using flow cytometry. Compared with the cells transfected with empty GC vesicles (Fig. 6A), following treatment with 10 µM RA, the percentage of cells in the G₀/G₁ phase significantly increased in the U118 and U138 cell lines, and the percentage of cells in the S and G₂/M phases subsequently decreased, (Fig. 6). The increase in the concentration of RA to 25 µM led to a further increase in the percentage of cells in the G₀/G₁ phase, with a subsequent decrease in the percentage of cells in the S and G₂/M phases (Fig. 6). These results findings suggested that RA transfection arrested the cell cycle in the G₀/G₁ phase in the human glioma cell lines.