Saponin 6 (>98% purity) was provided by Professor Tang (Institute of Materia Medica, School of Pharmacy, Fourth Military Medical University, Xi'an, China). The purity of saponin 6 was determined using a Dionex P680 high pressure liquid chromatography pump (Dionex; Thermo Fisher Scientific, Inc., Waltham, MA, USA) equipped with a UV 70 UV/Vis detector at 206 nm, and a YMC-Pack R&D ODS-A column (YMC Co., Ltd., Kyoto, Japan). Saponin 6 was dissolved in dimethyl sulfoxide (DMSO; Thermo Fisher Scientific, Inc.) and diluted with water to generate the 111.61 µM stock solution, containing no more than 0.1% DMSO. The stock solution was stored in aliquots at −20°C. Prior to experimentation, the stock solution was thawed, sterilized by filtration through a 0.22-µm sterile filter (EMD Millipore, Billerica, MA, USA), and diluted in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) containing 0.1% DMSO. DMEM containing 0.1% DMSO was used as the vehicle control in subsequent experiments.

The human U87 MG cell line and the murine HT-22 hippocampal neuronal cell line were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in DMEM supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified incubator containing 5% CO₂.

Cell viability was determined using the Cell Counting kit 8 (CCK-8) assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) (12,13). Briefly, U87 MG and HT-22 cells were seeded in 96-well plates (2×10⁴ cells/well) and incubated overnight. Subsequently, the cells were treated with saponin 6 at various concentrations (vehicle control, 1.79, 3.57, 7.14 and 14.28 µM) for 24 or 48 h. The optical density was measured at 450 nm using a Model 680 microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

DNA fragmentation was assessed using TUNEL staining (Roche Molecular Diagnostics, Pleasanton, CA, USA). Briefly, U87 MG cells were seeded in 24-well plates (1×10⁵ cells/well) and incubated overnight. Subsequently, the cells were treated with 2.83 or 5.66 µM saponin 6 for 24 h, washed with phosphate-buffered saline (PBS), and fixed in 4% paraformaldehyde at 4°C for 10 min. Following two PBS washes, the cells were permeabilized by incubating with 0.1% sodium citrate/0.1% Triton X-100 for 20 min. The cells were then washed again and incubated with the TUNEL reaction mixture at 37°C for 60 min. Cell nuclei were counterstained with Hoechst 33342 (Beyotime Institute of Biotechnology, Haimen, China). Cell morphology was examined under a fluorescence microscope (FV-1000; Olympus Corporation, Tokyo, Japan). Images of the cells were acquired using a Q-imaging video camera (QImaging, Surrey, BC, Canada).

Cell apoptosis was determined by flow cytometry using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining. Briefly, U87 MG cells were seeded in 24-well plates (2×10⁵ cells/well) and incubated overnight. Subsequently, the cells were treated with vehicle only, 2.83 µM or 5.66 µM saponin 6 for 24 h. Following the treatment, the cells were harvested by trypsinization, washed with cold PBS, and double-stained with Annexin V-FITC and PI (Immunotech S.A., Marseilles, France) at room temperature for 15 min. The cells were immediately subjected to flow cytometric analysis using a FACSCalibur™ system (BD Biosciences, Franklin Lakes, NJ, USA). Data were processed using ModFit software version 3.0 (Verity Software House, Topsham, ME, USA) and presented in a quadrantal diagram.

U87 MG cells were seeded in culture dishes (2×10⁵ cells/ml) and incubated overnight. Subsequently, the cells were treated with vehicle only, 2.83 or 5.66 µM saponin 6 for 24 h. The cells were harvested by trypsinization and fixed with 2.5% glutaraldehyde (PBS, pH 7.4) at 4°C overnight. The cells were then post-fixed in 1% buffered osmium tetroxide at room temperature for 2 h, dehydrated through a graded series of ethanol solutions, and embedded in Poly/Bed (Polysciences, Warrington, PA, USA) for 24 h at 60°C. Ultrathin sections (70–90 nm) were cut on an ultramicrotome and double-stained with uranyl acetate and lead citrate. The cell TEM micrographs were acquired using a JEM-2000EX electron microscope (JEOL, Ltd., Tokyo, Japan).

U87 MG cells were seeded in 6-well plates (2×10⁵ cells/ml) and incubated overnight. Subsequently, the cells were treated with vehicle only, 2.85 µM or 5.66 µM saponin 6 for 24 h. Following the treatment, the cells were collected by trypsinization, washed in cold PBS, and fixed in 75% ethanol at 4°C overnight. The fixed cells were subsequently washed with cold PBS and incubated with 50 µg/ml PI solution containing 50 µg/ml RNase A (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 30 min in the dark. Cell cycle analysis was performed using a flow cytometer (FACSCalibur™; BD Biosciences) and ModFit software (Verity Software House).

U87 MG cells were treated with vehicle only, 2.83 µM or 5.66 µM saponin 6 for 24 h as previously described. The cells were then lysed in radioimmunoprecipitation assay buffer [150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris-HCl, pH 8.0] supplemented with a complete protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) at 4°C for 30 min. The cell lysates were centrifuged at 15,000 × g for 15 min, and the supernatants were collected. The protein concentrations were determined using a Bicinchoninic Acid Protein Assay kit (Thermo Fisher Scientific, Inc.). Protein samples (25 µg) were then separated by 12% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (EMD Millipore). Subsequent to blocking the membranes in 5% nonfat dry milk containing 0.1% Tween-20 (Sigma-Aldrich) for 2 h, the membranes were incubated with polyclonal rabbit anti-Fas (1:500; cat. no. BS1461; Bioworld Technology, Inc., St. Louis Park, MN, USA), polyclonal rabbit anti-Fas ligand (FasL; 1:500; cat. no. BS1122; Bioworld Technology, Inc.), poly-clonal rabbit anti-caspase-3 (1:1,000; cat. no. ab90437; Abcam, Cambridge, MA, USA), monoclonal rabbit anti-caspase-8 (1:800; cat. no. 9496; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-caspase-9 (1:500; cat. no. BS1388; Bioworld Technology, Inc.), polyclonal rabbit anti-B-cell lymphoma 2 (Bcl-2; 1:500; cat. no. BS1511; Bioworld Technology, Inc.), polyclonal rabbit anti-Bcl-2-associated X protein (Bax; 1:500; cat. no. BS2538; Bioworld Technology, Inc.), and mouse monoclonal anti-β-actin (1:1,000; cat. no. sc-47778; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) antibodies at 4°C overnight. The membranes were subsequently washed three times with 0.1% Tween-20 in PBS (10 min each wash) and incubated with the secondary antibodies bovine anti-mouse IgG-horseradish peroxidase-conjugated (HRP) (cat. no. sc-2371) and bovine anti-rabbit IgG-HRP (cat. no. sc-2370) (both 1:2,000; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Immunoreactive bands were visualized using the enhanced chemiluminescence detection system (GE Healthcare, Uppsala, Sweden). Data were normalized to β-actin using ImageJ version 1.47 software (imagej.nih.gov/ij/).

All data are presented as the mean ± standard deviation calculated from at least three independent experiments. Data were analyzed using SPSS 20.0 (IBM SPSS, Armonk, NY, USA) or GraphPad Prism 5.01 (GraphPad Software, Inc., La Jolla, CA, USA) software. Differences between groups were analyzed using Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

The chemical structure and high-performance liquid chromatography (HPLC) chromatographs of saponin 6 are presented in Fig. 1A–C. Saponin 6 was >98% pure, as assessed by HPLC. The effects of saponin 6 were initially determined on the cell viability of human U87 MG cells and murine HT-22 hippocampal neuronal cells using the CCK-8 assay. Treatment with saponin 6 significantly decreased U87 MG cell viability in a time- and dose-dependent manner (Fig. 1D). The half maximal inhibitory concentration (IC₅₀) of saponin 6 was calculated as 2.83 µM following a 48 h treatment. However, low concentrations of saponin 6 (<3.57 µM) did not significantly affect the viability of HT-22 cells following treatment for 48 h. The IC₅₀ value of saponin 6 against HT-22 cells was 6.24 µM, which was more than two-fold higher than the value against U87 MG cells (Fig. 1E). These results indicate that saponin 6 may exhibit selective cytotoxicity against glioblastoma cells.

To further investigate saponin 6-induced U87 MG cell death, the present study assessed the effects of saponin 6 on DNA fragmentation, nuclear morphology and cell apoptosis using TUNEL staining, TEM and flow cytometry, respectively. TUNEL staining detected fragmented DNA, and the TEM micrographs revealed the loss of cellular microvilli and the presence of condensed, fractured and marginalized chromatin following a 24 h treatment with saponin 6. In addition, these effects increased with the increasing concentration of saponin 6 (Fig. 2A). Flow cytometric analysis using Annexin V-FITC/PI double staining revealed that saponin 6 significantly increased the number of early and late apoptotic cells in a dose-dependent manner (Fig. 2B and C). In particular, ~70% of cells underwent apoptosis (early or late stage) following a 24 h treatment with 5.66 µM saponin 6 (Fig. 2D). Furthermore, Hoechst 33342 staining revealed the presence of pyknotic nuclei and apoptotic bodies in TUNEL-positive cells (Fig. 2E), further confirming the activation of apoptosis. Although treatment with saponin 6 also increased cell necrosis, this accounted for only a small fraction of cell death induced by saponin 6 (Fig. 2B and C). These results indicate that saponin 6 may induce U87 MG cell death, predominantly via the promotion of cell apoptosis.

To fully characterize the effects of saponin 6, cell cycle progression of the U87 MG cells was determined by flow cytometry using PI staining. Treatment with saponin 6 significantly increased the percentage of cells at G₀/G₁ phase, and decreased the percentage of cells at S phase in a concentration-dependent manner (Fig. 3). These results indicate that saponin 6 may induce G₀/G₁ arrest in U87 MG cells.

To explore the signaling pathways underlying saponin 6-induced cell apoptosis in U87 MG cells, the effects of saponin 6 were determined on the protein expression levels of: The Fas death receptor and its ligand FasL; proteins involved in the extrinsic (death receptor) apoptotic pathway; Bcl-2 family proteins, including Bax (pro-apoptotic) and Bcl-2 (anti-apoptotic); and key regulators of the intrinsic (mitochondrial) apoptotic pathway. Treatment with saponin 6 for 24 h significantly increased the expression levels of Fas and FasL, and decreased Bcl-2 expression in a dose-dependent manner; however, there were no significant effects on Bax expression (Fig. 4A). These results suggest that saponin 6 may induce U87 MG cell apoptosis via activation of both the extrinsic and intrinsic apoptotic pathways. In addition, the protein expression levels of cleaved caspase-8 (an initiator caspase in the extrinsic pathway), cleaved caspase-9 (an initiator caspase in the intrinsic pathway), and cleaved caspase-3 (an effector caspase in both the extrinsic and intrinsic pathways) were detected. Treatment with saponin 6 significantly increased the expression levels of cleaved caspase-8, -9 and -3 in a concentration-dependent manner (Fig. 4B–C). These results are in agreement with the conclusion that saponin 6 may activate both the extrinsic and intrinsic apoptotic pathways in U87 MG cells.