SSd (purity, 96%) was purchased from the National Institutes for Food and Drug Control of China (Beijing, China). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) was obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Dulbecco's modified Eagle's medium (DMEM) was purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Akt (cat. no. 4691), phosphorylated (p)-Akt (cat. no. 4060), extracellular signal-regulated kinases (ERK; cat. no. 4695), p-ERK (cat. no. 4370), c-Jun N-terminal kinases (JNK; cat. no. 9258), p-JNK (cat. no. 4668), cleaved caspase-3 (cat. no. 9664), β-actin (cat. no. 12620) and peroxidase-conjugated anti-rabbit (cat. no. 7074) antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Chemicals for buffer preparations were purchased from Sigma-Aldrich (Merck KGaA).

Human U87 glioma cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and grown in DMEM containing 10% fetal bovine serum (Haoyang Biological Products Technology Co., Ltd., Tianjin, China), 100 U/ml penicillin, 100 µg/ml streptomycin, and incubated in a humidified atmosphere of 5% CO₂ at 37°C.

Cell viability was assessed using an MTT bromide assay. U87 cells (200 µl; 3×104 cells/ml) were seeded into 96-well tissue culture plates. Following overnight incubation, the cells were exposed to serial concentrations of SSd (1, 2, 3, 4, 5, 6, 7 and 8 µM) or control medium for 48 h. Subsequently, 10 µl MTT reagents was added to each well and incubated at 37°C for 4 h, followed by the addition of 150 µl dimethyl sulfoxide to each well. The plates were agitated for 10 min. Then, the absorbance was read at a wavelength of 490 nm using an iMark™ microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Each experiment was performed with six replicate wells for each condition, and the data were obtained from three independent experiments. The half-maximal inhibitory concentration (IC₅₀) of SSd was calculated using the Logit method (15).

U87 cells in 6-well plates were treated with different concentrations of SSd for 48 h, washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 10 min. Subsequently, the cells were washed with PBS, and stained with Hoechst 33258 for 5 min at room temperature. The nuclear morphology was observed using a laser scanning confocal microscope. For quantification, three different fields were randomly selected and counted under the microscope. The apoptotic rates were calculated as the percentage of apoptotic cells relative to the total number of cells.

Cells treated with different concentrations of SSd were seeded into 6-well plates (1.8×105 cells/well) for 48 h and isolated with trypsin. The cells were supplemented with 100 µl DMEM medium and analyzed according to the manufacturer's instructions using the Muse™ Annexin V and Dead Cell Assay kit (Muse™ Cell Analyzer; Merck KGaA).

Following SSd treatment for 48 h, U87 cells were washed with ice-cold PBS three times and resuspended in 100 µl radioimmune precipitation buffer [100 mM NaCl and 100 mM sodium fluoride, 20 mM Tris-HCl (pH 7.4), 2.5 mM EDTA, 1% SDS, 1% Triton X-100 and 1% sodium deoxycholate]. The lysate was centrifuged at 12,000 × g for 20 min, and 50 µg cell lysate protein was used for western blotting. Proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were incubated overnight individually with primary antibodies at a dilution of 1:1,000 in TBS containing 0.1% Tween-20 at 4°C, and the membranes were incubated with peroxidase-conjugated anti-rabbit IgG (1:5,000) for 2 h at room temperature. All bands were detected using the enhanced chemiluminescence (ECL) system (Tanon Science & Technology Co., Ltd., Shanghai, China) according to the manufacturer's instructions (16).

The data are presented as the mean ± standard deviations as indicated. Statistical analyses for comparison of mean values were performed by one-way analysis of variance followed by Dunnett's test. P<0.05 was considered to indicate a statistically significant difference. All data were derived from three independent experiments.

The mitochondria of living cells break down MTT to produce formazan, and the quantity of formazan corresponds with the living cell number. To determine whether SSd inhibits U87 glioma cell growth, the cell viability rate was assessed using the MTT method. In the present study, obvious inhibitory effects on the proliferation of U87 cells were observed with SSd exposure. As shown in Fig. 1, following treatment with 1–8 µM SSd for 48 h, the proliferation rate of U87 cells was significantly reduced in a dose-dependent manner when compared with the control group. Additionally, the half maximal inhibitory concentration (IC₅₀) value of SSd was 4.79 µM. The result indicates that SSd inhibited the viability of U87 glioma cells.

When detecting nuclear morphological changes, U87 cells were stained with Hoechst 33258 and observed under a laser scanning confocal microscope. Intact nuclei in the living cells were stained blue, and the morphology was round or oval, while the condensed or fragmented nuclei in apoptotic cells were stained bright blue. As illustrated in Fig. 2, the cells treated with 2.5 µM SSd began to exhibit nuclear morphological changes when compared with the control cells, and the nuclei of cells treated with 7.5 µM SSd were markedly brighter, indicating a high prevalence of nuclear chromatin and fragmentation.

To further evaluate whether the inhibition of cell proliferation in U87 cells was associated with the induction of apoptosis, Annexin V staining was used to assess the rate of apoptotic cells following SSd treatment. As shown in Fig. 3, treatment with SSd significantly increased the proportion of Annexin V-positive cells in a dose-dependent manner. The results indicate that SSd treatment inhibited the viability of U87 cells by inducing apoptosis.

To further elucidate the possible mechanisms underlying the effects of SSd treatment on U87 cells, the p-Akt/Akt, p-ERK/ERK, p-JNK/JNK protein expression levels with western blot analysis. The data demonstrated that SSd exposure induced a significant decrease of p-Akt and p-ERK relative protein expression levels, and increased p-JNK protein expression levels (Fig. 4; P<0.05). These results indicated that treatment with SSd potentially downregulated the PI3K/Akt and ERK signaling pathways, increased JNK activation and further enhanced apoptosis in U87 cells.

Caspases are critical mediators during the process of apoptosis. In all of apoptosis-associated caspases, caspase-3 is a key effector or executioner for programmed cell death (17). Thus, to determine whether caspase-3 was involved in SSd-induced cytotoxicity, the expression levels of cleaved caspase-3, an indicator of its activity, were examined. In the present study, SSd treatment significantly increased the expression level of cleaved caspase-3 (Fig. 5), indicating that SSd-induced apoptosis in U87 cells may be associated with the promoted activation of caspase-3.