Primary mouse cortical neurons were prepared as described previously (18). In brief, cortical neuronal cells were isolated from embryos of time-mated pregnant mice and cultured onto poly-D-lysine-coated 96-well dishes at a density of 2×10⁴ cells/well in neurobasal medium (Life Technologies, USA) supplemented with 2% B27 (Life Technologies) and 1 mM glutamate (G3291; Sigma-Aldrich). Cells were maintained in growth medium at 37°C in 5% CO₂. Half of the culture medium was replaced with fresh medium every 3 days. All experiments were performed after 10–12 days in vitro. Human U251MG, U87MG and A172 glioblastoma cell lines were obtained from the American Tissue Culture Collection (ATCC; Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, USA) with 10% fetal bovine serum (FBS; 10099141, Life Technologies) and 1% penicillin/streptomycin at 37°C in a 5% CO₂ incubator. The human SHG44 glioblastoma cell line originally present in our laboratory was cultured in RPMI-1640 (Life Technologies) medium with 10% FBS and 1% penicillin/streptomycin at 37°C in a 5% CO₂ incubator.

Wogonoside was purchased from Shanghai Tauto Biotech Co., Ltd. (Shanghai, China). U0126, SB203580, SP600125, LY294002, 3-methyladenine (3-MA), bafilomycin A (BafA1), N-acetyl-cysteine (NAC) and acridine orange (AO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Z-VAD-FMK (Z-VAD) was purchased from Abcam (Cambridge, MA, USA). Anti-LC3 and anti-Beclin 1 antibodies were purchased from Novus Biologicals (Littleton, CO, USA). Anti-cytochrome c, anti-p62/SQSTM1 and anti-Bcl-2-associated X protein (Bax) antibodies were purchased from Abcam. Anti-phosphorylated extracellular regulated protein kinase (ERK), anti-ERK, anti-phosphorylated c-Jun N-terminal kinase (p-JNK), anti-JNK, anti-phosphorylated p38, anti-p38, anti-phosphorylated AKT, anti-AKT, anti-phosphorylated mTOR, anti-mTOR, anti-phosphorylated p70S6K, anti-p70S6K, anti-poly(ADP-ribose) polymerase (PARP), anti-caspase-3, anti-caspase-8, anti-autophagy-related 3 (Atg3), anti-autophagy-related 5 (Atg5), anti-autophagy-related 7 (Atg7), anti-autophagy-related 12 (Atg12), anti-B-cell lymphoma 2 (Bcl-2), anti-β-actin and goat anti-rabbit IgG (H+L)-HRP secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Cell viability was assessed by the CCK-8 assay (Beyotime, Shanghai, China). For primary mouse cortical neurons, 2×10⁴ cells/well were seeded onto 96-well culture plates and cultivated for 10–12 days to adhere. For glioblastoma cells, 5×10³ cells/well were seeded onto 96-well culture plates and cultivated for 24 h to adhere. After treatment, CCK-8 (10 μl) was added into every well and incubated for 2 h, and the OD value was read at 450 nm using a Bio-Rad enzyme linked immunosorbent assay (ELISA) microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). The following formula was used: Cell viability = (OD of the experimental sample/OD of the control group) × 100%.

After treatment, cells were harvested and lysed. An equal amount of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 10%) and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% non-fat dried milk for 2 h, the membrane was incubated with the primary antibodies overnight at 4°C. Then the immunoreactive bands were visualized by enhanced chemiluminescence using HRP-conjugated IgG secondary antibodies.

Treated cells were fixed using 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and then blocked with immunostaining blocking buffer (Beyotime, Shanghai, China). Subsequently, cells were incubated with the LC3 antibody overnight at 4°C. Following washes with phosphate-buffered saline (PBS), the coverslips were incubated with a red-labeled secondary antibody (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China) for 4 h at room temperature. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and then cells were washed with PBS and mounted with anti-fade medium. The staining was examined using a fluorescence microscope.

After treatment, cells were washed twice with PBS and collected by trypsinization. After centrifugation at 1000 × g for 5 min at room temperature, the cells were re-suspended and stained with Annexin V and propidium iodide (PI) (BD Biosciences, San Jose, CA, USA) for 15 min at room temperature in the dark. Flow cytometry was performed using a FACScan laser flow cytometer (FACSCalibur; Becton Dickinson). Then the apoptotic rate was analyzed by the FACScan laser flow cytometer (FACSCalibur).

Treated cells were stained with 1 μg/ml AO for 15 min at 37°C. After incubation, cells were washed three times with PBS and then observed immediately under a fluorescence microscope.

After treatment, cells were collected by trypsinization, washed three times with warm PBS (37°C) and then fixed for 1 h in 2.5% glutaraldehyde in 0.1 M cacodylate buffer with 1% sucrose. After washing in PBS, cells were then embedded, sectioned, double stained with uranyl acetate and lead citrate (Fluka, St. Louis, MO, USA) and viewed using a JEOL JEM-1011 TEM (Tokyo, Japan).

The lentiviral vectors were purchased from GenePharma Co., Ltd. (Shanghai, China). Lentiviral vectors for expression of scrambled or Beclin 1 shRNA were diluted in medium containing 5 μg/ml Polybrene. The shRNAs were then added to the cultures. After 72 h, the transfected cells were selected using puromycin (5 μg/ml) for 24 h. The human Beclin 1 shRNA sequence was 5′-GAA TGT CAG AAC TAC AAA CGC TGT T-3′, while the scrambled shRNA sequence was 5′-TTC TCC GAA CGT GTC ACG T-3′. Western blotting was performed to validate the knockdown efficiency, and cells were split for the different assays.

The SPSS19.0 software package was used to perform all statistical analyses. Data are expressed as means ± SD. The statistical significance of the differences between treatments was assessed using one-way ANOVA followed by Student-Neuman-Keuls test for multiple comparisons. A probability (P) value <0.05 was considered to indicate a statistically significant result.

To examine the effect of wogonoside on cell viability, four human glioblastoma cell lines, U251MG, SHG44, A172 and U87MG, were treated with different concentrations of wogonoside for different time periods. Cell viability assays showed that wogonoside induced cell death in both a concentration- and time-dependent manner in all the cell lines tested (Fig. 1). The IC₅₀ values of these cell lines are shown in Table I. Importantly, wogonoside alone did not affect the cell viability of the mouse primary neurons (Fig. 1A). Since U251MG was the most sensitive cell line in respond to wogonoside, this cell line was chosen for the subsequent studies unless otherwise indicated.

To determine whether the reduced cell viability was due to apoptosis, flow cytometric analysis for Annexin V/PI was used. As shown in Fig. 2A, a significant increase in the apoptotic population was observed in cells treated with wogonoside. In addition, wogonoside cleaved caspase-3 and PARP, downregulated Bcl-2, upregulated Bax and released cytochrome c from the mitochondria without affecting caspase-8 (Fig. 2B and C), suggesting that mitochondrial apoptosis was induced by wogonoside. To further demonstrate that wogonoside induced caspase-dependent apoptosis, cells were co-treated with wogonoside and the pan-caspase inhibitor Z-VAD, and the cell viability as well as caspase-3 and PARP cleavage were determined. The results revealed that Z-VAD treatment suppressed cell death and caspase-3 and PARP cleavage induced by wogonoside (Fig. 2D and E). Collectively, these data indicate that wogonoside induced cell death in human glioblastoma cells through the mitochondrial apoptosis pathway.

A previous study showed that wogonoside activates autophagy in breast cancer cells (7). Thus, we aimed to ascertain whether autophagy occurs in the cells treated with wogonoside. We first used immunofluorescence to clarify the ability of wogonoside to induce autophagy. Results of the fluorescence microscopy revealed that wogonoside induced the formation of autophagosomes, as indicated by the formation of LC3 punctate dots in the cells (Fig. 3A). Furthermore, TEM analysis showing autophagic vacuoles containing cellular material or membranous structures further confirmed the presence of autophagy (Fig. 3B). Meanwhile, wogonoside treatment increased the expression levels of Beclin 1, LC3-II and various other autophagy-related proteins in a time-dependent manner (Fig. 3C).

Induction of autophagy includes not only an increase in autophagosomes, but also an upregulation of autophagic flux. To examine the autophagic flux, we first analyzed the formation of AVOs by AO staining. As shown in Fig. 3D, treatment with wogonoside resulted in the formation of red fluorescent AVOs. During the process of autophagy, p62 is wrapped into autophagosomes and degraded in autolysosomes. Thus, we determined the protein levels of p62 in the wogonoside-treated cells. Results of the western blot analysis showed that wogonoside reduced the amount of p62 in a time-dependent manner (Fig. 3E). We further examined the autophagic flux by LC3 turnover assay. Treatment with BafA1, which blocks autolysosome formation, led to a significant increase in LC3-II levels. The BafA1-induced increase in LC3-II was augmented when combined with wogonoside (Fig. 3F). In line with the enhanced LC3 turnover, the wogonoside-induced reduction in p62 was prevented by BafA1 (Fig. 3F). Together, these data iindicate that wogonoside induced autophagy in glioblastoma cells.

We next aimed to elucidate the role of autophagy in wogonoside-induced apoptosis. To this end, we first used 3-MA, an inhibitor that blocks the autophagy pathway at early stages. As shown in Fig. 4A–C, 3-MA reversed not only autophagy but also the cell death and apoptosis caused by wogonoside. To exclude the off-target effects of 3-MA, we used shRNA to specifically knock down Beclin 1. Fig. 4D shows that the levels of Beclin 1 and LC3-II were apparently decreased in the Beclin 1 shRNA-treated cells. In addition, knockdown of Beclin 1 decreased cell death and cleavage of PARP (Fig. 4E and F). Finally, we used flow cytometry to determine the role of autophagy in apoptosis. As shown in Fig. 4G, both 3-MA and knockdown of Beclin 1 reduced the apoptosis in response to wogonoside. Collectively, these data strongly suggest that wogonoside-induced autophagy is a prerequisite to cell apoptosis.

It has been well documented that the MAPK and PI3K/AKT/mTOR/p70S6K signaling pathways play an important role in regulating both autophagy and apoptosis. Therefore, the effects of wogonoside on the activity of these two pathways were studied. Fig. 5A and B showed that wogonoside treatment significantly increased the phosphorylation of p38 and decreased the phosphorylation of AKT, mTOR and p70S6K in a time-dependent manner. These results suggest that p38 MAPK and PI3K/AKT/mTOR/p70S6K signaling pathways may participate in the autophagy and apoptosis induced by wogonoside. To confirm this, we assessed the effects of a number of pharmacologic inhibitors, including those of MEK1/2 kinase (U0126), JNK kinase (SP600125), p38 kinase (SB203580) and PI3K kinase (LY294002) on the cell death induced by wogonoside. Of these inhibitors, SB203580 exhibited the ability to counteract the cell killing activity of wogonoside while LY294002 showed the opposite effect (Fig. 5C). Furthermore, western blotting and flow cytometric analysis confirmed that p38 MAPK and PI3K/AKT/mTOR/p70S6K signaling pathways were involved in wogonoside-induced apoptosis and autophagy (Fig. 5D–H). Taken together, these results indicate that autophagy and apoptosis induction by wogonoside were mediated partly through the p38 MAPK and PI3K/AKT/mTOR/p70S6K signaling pathways.

Recently, ROS were reported to be associated with autophagy and apoptosis induced by antineoplastic drugs (19,20). Similarly, we found that pretreatment of cells with the antioxidant NAC rescued wogonoside-induced cell death, apoptosis and autophagy (Fig. 6A and B). All together, these results indicate that ROS are mediators between wogonoside and autophagy and apoptosis.

To prove that the apoptosis and autophagy induced by wogonoside were not cell-specific, we examined the anticancer effect of wogonoside on another human glioblastoma cell line, U87MG. We found that wogonoside induced both apoptosis and autophagy in the U87MG cells (Fig. 7A and B). Z-VAD, 3-MA and knockdown of Beclin 1 rescued the cell death induced by wogonoside (Fig. 7C). Wogonoside also activated p38 MAPK and inhibited AKT (Fig. 7D). Furthermore, SB203580 and NAC reduced wogonoside-triggered cell death while LY294002 aggravated it (Fig. 7E). Similar results from these two glioblastoma cell lines indicated that autophagy induced by wogonoside promoted apoptosis, and that wogonoside-induced autophagy and apoptosis were due to the activation of the p38 MAPK signaling pathway, inhibition of the PI3K/AKT/mTOR/p70S6K signaling pathway and generation of ROS.