The C6 rat glioblastoma cell line was purchased from American Type Culture Collection (Rockville, MD, USA). The cells were cultured under sterile conditions at 37°C in a humid environment with 5% of CO₂ in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and 1% antibiotics and antimycotics (all Welgene, Daegu, Korea).

Abeliophyllum distichum Nakai [voucher no. Park1001(ANH)] was collected in Misunhyang Theme park, Seongbul-Mountain Recreation Forest, 78, Chungmin-rogigok-gil, Goesan-eup, Goesan-gun, Chungcheongbuk-do, Korea.

To induce callus formation (27), 1-cm² leaf explants were isolated from the fresh plants. The explant was cultured on Murachige and Skoog medium (4% sucrose, 0.9% agar supplemented with 1 mg/l naphthalene acetic acid and 1 mg/l 2,4-dichlorophenoxyacetic acid, pH 5.7) at 25°C. The callus was induced 20 days later. A sufficient quantity of acteoside was obtained through subculture to separate and purify acteoside from the callus (Fig. 1). Different batches of purified acteoside were used in each experiment. Each experiment was performed three times using a different batch.

The ethylacetate fractions were concentrated in vacuo and used as a sample for the purification of acteoside. Acteoside was isolated and purified by the Accelerated Chromatographic Isolation system (Isolera™ Spektra, Biotage, Uppsala, Sweden) using SNAP KPHOSPHO-SIL and SNAP Ultra Cartridges (Biotage). The acteoside purified from the callus was quantitatively analyzed using the standard acteoside by HPLC-PDA analysis. Finally, acteoside of ≥95% purity was obtained from the callus and used as a sample for chemotherapy of temozolomide-based glioblastoma.

To identify the TMZ half maximal inhibitory concentration (IC₅₀ value) against C6 cells, 1×10⁴ cells in single cell suspensions were seeded into individual wells of 96-well plates and incubated for 24 h at 37°C prior to TMZ treatment at the indicated concentrations (1, 5, 10, and 20 mM) at 37°C for 24 h. Following determination of the TMZ IC₅₀ value as 5 mM, the cells were treated for 24 h with 5 mM TMZ, 50 µM acteoside, or a combination of the two (5 mM TMZ and 50 µM acteoside). MTT solution (5 mg/ml) was added to each well (10 µl) and cultured at 37°C for 2 h. Subsequently, the medium was removed and DMSO was added at a volume of 200 µl each and reacted at room temperature for 30 min. The absorbance at 595 nm was measured to determine cell viability.

A C6 cell suspension in 1 ml was cultured in a 12-well plate and grown to confluence. The cells were treated with TMZ, acteoside or TMZ + acteoside and then scratched with a sterile 10-µl pipette tip to create an artificial wound. At 0 and 24 h post-wounding, digital images of the wound healing process were captured using an inverted microscope. Cell migration was quantified by measuring the size of the scar at 0 and 24 h using the image analyzing software, ImageJ 1.43u/Java1.6.0_22 software (NIH, Bethesda, Maryland, USA). Each experiment was performed three times and the measurements were performed in triplicate.

The C6 cells were treated with TMZ, acteoside or TMZ + acteoside for 24 h. Cells were lysed on ice by the RIPA (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS) lysis buffer supplemented with protease and phosphatase inhibitors cocktail (Roche, Basel, Switzerland) for 30 min. After centrifugation at 18,341 × g for 15 min at 4°C, supernatant was obtained. Protein concentration was determined using Bradford Protein Assays (Bio-rad, Hercules, CA, USA). Equal amounts of total proteins (30 µg) were loaded into single wells and fractionated via electrophoresis via a 10-15% SDS-PAGE. The proteins were electrotransferred to polyvinylidene difluoride membranes (EMD Millipore, Bedford, MA, USA). Following incubation in blocking solution containing 5% nonfat milk in Tween/Tris-buffer saline for 30 min at room temperature. The blots were probed with 1:1,000-diluted primary antibodies (cat. no. 9662, caspase 3; Cell Signaling Technology, Inc., Danvers, MA, USA), B0-cell lymphoma 2 (sc-7382, Bcl-2), Bcl-2-associated X protein (cat. no. 2772, Bax), phosphorylated (p-)p53 (sc-101762), total-p53 (sc-6243), β-actin (cat. no. 4970; all; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), LC-3 (L8918, Sigma; Merck KGaA, Darmstadt, Germany), Rab7 (cat. no. 9367, Cell Signaling Technology, Inc.), p62 (cat. no. 114), p-p38 (cat. no. 9211), total-p38 (cat. no. 9212), p-c-Jun N-terminal kinase (cat. no. 9251, p-JNK), total-JNK (cat. no. 9252), p-extracellular signal-regulated kinase (cat. no. 9101, p-ERK), total-ERK (cat. no. 9102; all Cell Signaling Technology, Inc.) overnight at 4°C. This was followed by incubation with horseradish peroxidase-conjugated secondary antibodies (1:2,000; LF-SA8001A, Goat Anti-Mouse IgG-HRP) or LF-SA8002A (Goat Anti-Rabbit IgG-HRP), AB Frontier, Seoul, Korea.) for 2 h at room temperature. The proteins were then visualized by exposure to Chemi-Doc (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Lysosomal-associated membrane protein 1 (LAMP1) and LC3 are markers for lysosomal and autophagy. The cells (1×10⁵ cells/well) were prepared on sterilized glass coverslips (BD Biosciences, Franklin Lakes, NJ, USA) in triplicate. Following treatment with TMZ, acteoside or TMZ + acteoside for 24 h, the cells were fixed in 4% paraformaldehyde for 30 min, blocked with 5% normal chicken serum (S-3000; Vector Laboratories, Inc., Burlingame, CA, USA), 0.1% Triton X-100 and then incubated 1 h for permeabilization and to block non-specific protein-protein interactions. The cells were then incubated with the anti-LAMP1 (1:200; sc-19992, Santa Cruz Biotechnology, Inc.) and anti-LC3 (1:400; PM036, MBL International, Woburn, MA, USA) overnight at 4°C. The cells were then washed twice with PBS and incubated for an additional 2 h in the dark with a mixture of Alexa Fluor 488 and 594 secondary antibodies (1:200; Alexa Fluor 488 (A-11008) and 594 (A-11007), Thermo Fisher Scientific, Inc., Waltham, MA, USA) and washed again with PBS. The nuclei were stained using 4,6-diamidino-2-phenylindole (Sigma; Merck KGaA), and then washed twice with PBS. The slides were then mounted and images were captured under an LSM-700 laser confocal microscope.

The cells were analyzed for Mitotracker Green and MitoSOX by flow cytometry using a FACSCanto II flow cytometer, as indicated by the manufacturer (BD Biosciences). Following two washes with PBS, the cells were fixed in 4% paraformaldehyde for 30 min at room temperature and permeabilized with 0.25% Triton X-100 in PBS for 20 min. The cells were stained with primary antibodies for overnight at 4°C (1:200) and then with secondary antibodies for 1 h on ice. Following two washes with PBS, the cells were fixed in 4% paraformaldehyde and assayed immediately. The flow cytometry data were collected using 10,000 cells and were analyzed using FlowJo 7.6.1 software (Tree Star, Inc., Ashland, OR, USA).

All data were analyzed using GraphPad Prism 5.0 and are presented as the mean ± standard error of the mean. Data were analyzed with one-way analysis of variance with Tukey's multiple-comparisons post hoc test for the comparison of mean values among multiple groups. P<0.05 was considered to indicate a statistically significant difference.

Combined treatment with TMZ and acteoside inhibited the viability of C6 cells. TMZ reduced cell viability in a dose-dependent manner (Fig. 2A), whereas acteoside alone had no significant effect at any treatment level (Fig. 2B). Following incubation in culture medium containing TMZ with or without acteoside for 24 h, an MTT assay was performed to compare the cytotoxicities of different treatment levels. TMZ significantly suppressed cell viability, whereas acteoside did not significantly suppress cell growth. Combined treatment with TMZ and acteoside markedly reduced cell viability (Fig. 2C). Treatment with TMZ or acteoside applied alone reduced wound-healing ability (Fig. 3A). The wound distance (%) was measured using the results shown in Fig. 3A with ImageJ software. The wound distance increased significantly following combination treatment (Fig. 3B), compared with either individual treatment. These results indicate that cotreatment with TMZ and acteoside was an effective inhibitor of glioblastoma cell proliferation and migration.

The results of the western blot analysis revealed that the levels of cleaved caspase-3, Bax and phosphorylated p53 were higher and the levels of Bcl-2 were lower following combination treatment with TMZ and acteoside than following treatment with TMZ alone (Fig. 4A). Acteoside-mediated mitochondrial function and the generation of reactive oxygen species (ROS), which are key mediators of apoptotic signaling, were then observed in the TMZ-treated C6 cells. To verify mitochondrial mass, fluorescence-activated cell sorting analysis was performed using MitoTracker Green. Cotreatment with TMZ and acteoside resulted in a higher mitochondrial mass than treatment with TMZ alone (Fig. 4B). ROS generation was significantly higher following cotreatment with TMZ and acteoside than treatment with TMZ alone (Fig. 4C). These results suggest that ROS generation and mitochondrial function are important in apoptosis induced by treatment with TMZ + acteoside.

TMZ has been reported to induce autophagy (28,29); therefore, the present study examined the extent to which autophagy was induced by TMZ + acteoside treatment. The C6 cells were treated with TMZ with or without acteoside; after 24 h, and the cells were stained for immunofluorescence to detect LAMP1 and LC3 as markers of autophagy. Higher expression levels of LAMP1 and LC3 were observed following the combination treatment (Fig. 5A). Autophagy-related protein expression was also examined, and it was found that TMZ induced the conversion of LC3-I to LC3-II, which is a signature of autophagosome generation. A higher rate of conversion of LC3-I to LC3-II was observed following cotreatment with TMZ and acteoside than treatment with TMZ alone. The expression levels of Rab7 and p62 indicated autophagy induction in the TMZ-treated cells (Fig. 5B). These findings suggest that cotreatment with TMZ and acteoside enhanced the autophagic process in glioblastoma cells.

Previous studies have demonstrated that TMZ regulates the MAPK pathway, including p38, JNK and ERK (30). Therefore, the present study investigated whether acteoside affects TMZ-related MAPK gene expression. The C6 cells were treated with TMZ with or without acteoside. After 24 h, TMZ treatment resulted in higher expression levels of p-p38, p-JNK and p-ERK. The expression of TMZ-regulated genes was significantly higher following TMZ + acteoside treatment than treatment with TMZ alone (Fig. 6). These observations suggest that acteoside affects TMZ-based therapeutic mechanisms via the MAPK pathway.