The human GBM DBTRG-05MG cell line was maintained in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum, 0.01 M HEPES and 1 mM sodium pyruvate in a 37°C incubator with 5% CO₂.

Honokiol was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 50 mM and stored at −20°C. Sulforhodamine B (SRB) was dissolved in phosphate buffer at a concentration of 5 mg/ml and stored at 4°C. SRB, DMSO and cadmium acetate were purchased from Sigma (St. Louis, MO, USA).

The DBTRG-05MG cells were seeded at a density of 3×10³ cells/well in 96-well plates for 24 h. The cells were then treated with various concentrations of honokiol (6.25, 12.5, 25 and 50 μM) for 72 h. The cell numbers were determined using the SRB assay. Briefly, the cells were fixed in 10% trichloroacetic acid and stained with 0.4% SRB, a protein binding dye. Following incubation and washing with 1% acetic acid, the bound SRB was dissolved in 10 mM unbuffered Tris base and the optical density was measured at 562 nm using a microtiter plate reader.

One day after being seeded in a 6-well plate (1×10⁵ cells/ml, 2 ml/well), the cells were incubated with various concentrations of honokiol (12.5, 25 and 50 μM) for 72 h. The control groups were treated with phosphate-buffered saline (PBS) only. Upon harvesting, the cells were fixed in 70% ice-cold ethanol and stored at −20°C. The cells were then washed twice with ice-cold PBS and incubated with RNase and the DNA intercalating dye, propidium iodide (50 μg/ml). The percentages of the sub-G₁ apoptotic population and cell cycle distribution were then analyzed using a flow cytometer (BD Biosciences, San Jose, CA, USA).

The cells were seeded at density of 1×10⁶ cells/dish in a 10-cm dish for 24 h. To prepare the total cell extract, the cells were harvested at 72 h following the treatment described previously, then washed, lysed with lysis buffer [50 mM Tris-HCl (pH 7.4) 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM Na₃VO₄, 1 mM NaF and 2% cocktail] and cleared by centrifugation at 12,000 × g for 30 min at 4°C. Briefly, the cell pellets were lysed with protein extraction solution and incubated at −20°C for 20 min. Next, the cell lysates were centrifuged at 15,000 × g for 5 min and the total protein was collected. The protein concentration was measured using a protein assay kit (Strong Biotech, Taipei, Taiwan). Total protein (20 μg) was separated on a 10% SDS-PAGE gel and transferred to a PVDF membrane. Non-specific binding was blocked using 5% skimmed milk. Primary antibodies to detect RB (sc-102), poly(ADP-ribose) polymerase (PARP; sc-7150) and Bcl-x (S/L; sc-8392) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Primary antibodies to phospho-RB (Ser807/811), Beclin-1 (#3738), LC3 (#4108) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; #2118) were purchased from Cell Signaling Technology (Beverly, MA, USA). The primary antibodies were detected using horseradish peroxidase (HRP)-conjugated anti-mouse, anti-rabbit or anti-goat secondary antibodies as appropriate, for 1 h at 25°C. The bound HRP-conjugated secondary antibodies were visualized using an Enhanced Chemiluminescence (ECL) Plus System (Millipore, Billerica, MA, USA).

The SRB assay revealed that honokiol inhibited the growth of the DBTRG-05MG cells in a dose-dependent manner. Following 72 h of treatment, the IC₅₀ of honokiol on the DBTRG-05MG cells was ~30 μM (Fig. 1). To investigate the apoptotic effects of honokiol on the GBM cells, the percentage of the apoptotic sub-G₁ fraction was analyzed using a flow cytometer. Following the treatment with honokiol for 72 h, the percentage of the apoptotic sub-G₁ fraction was 9.02, 11.48, 12.42 or 53.55% in the control and the cells treated with 12.5, 25 and 50 μM of honokiol, respectively (Fig. 2). A marked increase in the apoptotic fraction was not observed in the cells that were treated with ≤25 μM honokiol. When the dose increased to 50 μM, a massive sub-G₁ fraction was observed, indicating that apoptosis only occurred at higher doses of honokiol (Fig. 2).

To improve our understanding of the events that are involved in honokiol-induced apoptosis, the effects of honokiol on the Rb, PARP and Bcl-x (S/L) proteins, which are known to regulate cell apoptotic cascades, were examined. At a dose of 25 μM, honokiol markedly decreased the phospho-Rb level, but did not significantly affect the total Rb level (Fig. 3). However, honokiol significantly decreased the phospho- and total Rb proteins when the dose reached 50 μM. This phenomenon was paralleled with the high level of apoptosis induced by the higher dose (50 μM) of honokiol shown in Fig. 2. Consistent with this, the cleavage of PARP, an apoptotic marker, and anti-apoptotic protein Bcl-x (S/L) were only observed in the cells that were treated with 50 μM honokiol (Fig. 4).

To investigate if autophagy was involved in the honokiol-induced effects against GBM cells, the protein levels of two hallmarks of autophagy, namely Beclin-1 and MAP1LC3A (microtubule-associated protein 1A/1B light chain 3), were examined in the honokiol-treated cells. Honokiol markedly increased the levels of Beclin-1 and LC3-II at 50 μM (Fig. 5). Compared with the results that are shown in Figs. 2–4, as in the induction of apoptosis, autophagy was also markedly triggered by honokiol at a dose of 50 μM. The proautophagic effects of honokiol may play a crucial role in the anticancer effects against GBM cells.