Honokiol (MW 266.33, C₁₈H₁₈O₂, Fig. 1) was purchased from Wako Chemical (Wako, Japan). Fetal bovine serum (FBS) was purchased from Gibco-BRL (Rockville, MD). Anti-ICAM-1 and anti-VCAM-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Enhanced chemiluminescence (ECL) western blotting detection reagent was purchased from Amersham (Buckinghamshire, UK). All other chemicals, including endothelial cell growth supplements (ECGS) and heparin, were purchased from Sigma-Aldrich (St. Louis, MO).

Human umbilical vein endothelial cells (HUVECs) were obtained from Clonetics (San Diego, CA), grown in medium 199 supplemented with 20% FBS, 2 mM L-glutamine, 5 U/ml heparin, 100 IU/ml penicillin, 10 μg/ml streptomycin and 50 μg/ml ECGS and incubated in a humidified 5% CO₂ incubator. HUVECs were plated at a density of 1×10⁷ cells per 100 mm dish. Cells were used between passage numbers 3 and 6.

Cell viability was determined colorimetrically using the MTT assay. Cells in the exponential phase were seeded at 1×10⁴ cells per well in 24-well plates. After different treatments, 20 μl of 5 mg/ml MTT solution was added to each well (0.1 mg/well), and the wells were incubated for 4 h. The supernatants were aspirated, the formazan crystals in each well were dissolved in 200 μl of dimethyl sulfoxide for 30 min at 37°C, and the optical density at 570 nm was read on a microplate reader (Bio-Rad, Hercules, CA).

Cells were lysed in PRO-PREP protein extraction solution. The sample was centrifuged at 13000 rpm for 15 min at 4°C. Protein concentration was determined by the Bradford method. An equal volume of 2X SDS sample buffer (0.1 M Tris-Cl, 20% glycerol, 4% SDS, and 0.01% bromophenol blue) was added to an aliquot of the supernatant fraction from the lysates, and the samples were boiled for 5 min. Aliquots of 30 μg of protein were subjected to 10% SDS-polyacrylamide gel electrophoresis for 1 h 30 min at 110 V. The separated proteins were transferred to a PVDF membrane for 2 h at 20 mA with the SD Semi-dry Transfer Cell (Bio-Rad). The membranes were blocked with 5% nonfat milk in Tris-buffered saline (TBS) containing 0.05% Tween-20 (TBS-T) for 2 h at room temperature. Then, the membranes were incubated with primary antibodies in 5% skim milk in TBS-T overnight at 4°C, and the bound antibody was detected by horseradish peroxidase-conjugated anti-rabbit IgG. The membranes were washed and then developed using a western blotting Luminol Reagent system (Amersham).

HUVECs were seeded into two-well chamber slides 48 h before the experiments. T98G cells (3×10⁷) were incubated in an RPMI-1640 medium containing 2% FBS and 10 mg/ml of the fluorescent dye BCECF/AM (Boehringer, Mannheim, Germany) at 37°C for 30 min. Fluorescently labeled cells were pelleted and resuspended (7.5×10⁵ cells/ml) in medium 199 with 10 mM HEPES buffer (M199H). HUVECs were washed three times with M199H before the dye-loaded cells were added and incubated at 37°C. After 30 min, cell suspensions were withdrawn, and the HUVECs were gently washed with M199H. Fluorescent images were obtained using a high-resolution video camera (DXC-960MD; Sony) mounted on a BH-2 Olympus microscope (Melville, NY), and the immunoreactivity of these images was measured using SigmaGel 1.0 (Jandel Scientific, Germany). The analyses were repeated three times over the same region, and the results are the mean values of the three independent experiments.

The Matrigel invasion assay was performed as previously described (31). Briefly, T98G cells treated with honokiol were collected, and 2×10⁵ cells/insert in serum-free media were added to the Matrigel-coated upper chambers (8 μm pore size, Falcon). RPMI media containing 10% FBS was added to the lower chambers, and the invasion chambers were incubated for 24 h in a 37°C cell culture incubator. The noninvasive cells that remained on the upper surface of the insert membranes were removed by scrubbing. The cells on the lower insert membranes were stained with DAPI, and the cells were counted under the light microscope. Each sample was measured in triplicate, and each experiment was repeated three times.

Cells at the exponential phase were seeded at 1×10⁷ cells/well on a slide glass. The cells were treated with honokiol for 24 h at 37°C, washed with PBS and fixed by the addition of methanol. Apoptotic cells were identified by a TUNEL assay of nucleosomal DNA fragments using a commercially available In Situ Cell Death Detection Kit (Roche, Penzberg, Germany), according to the manufacturer’s protocol, with a minor modification.

Scanning densitometry was performed using an Image Master^® VDS (Pharmacia Biotech Inc., San Francisco, CA). All data are expressed as the mean ± SD of results from the number (n) of experiments. Differences between data sets were assessed by Student’s t-test. P<0.05 indicated a statistically significant difference.

Adhesion molecules such as ICAM-1 and VCAM-1 have been shown to be involved in cell-cell and cell-ECM interactions and are mechanistically important for the extravasation of cancer cells during metastasis (1,29). The adhesion of circulating tumor cells to the microvascular endothelium of organs at distant sites is an important step in blood-borne metastasis. Accordingly, we first examined the effect of honokiol on ICAM-1 and VCAM-1 expression after TNF-α-stimulation of HUVECs. The cells were pretreated with varying doses of honokiol (1, 5, 10, 20 or 50 μM) for 24 h and were then co-treated with TNF-α (10 ng/ml) for 6 h. The results showed that TNF-α increased both ICAM-1 and VCAM-1 expression. This increase was significantly suppressed by honokiol from 20 μM, or 5 μM, respectively, suggesting that honokiol regulates the TNF-α-induced expression of VCAM-1 more effectively than that of ICAM-1 (Fig. 2).

Following the study of the effect of honokiol on ICAM-1 and VCAM-1 expression after TNF-α stimulation, the effect of honokiol on the adhesion of cancer cells to HUVECs was investigated. Adhesion of T98G cells to HUVECs stimulated with TNF-α at 10 ng/ml for 6 h was dramatically increased compared to unactivated HUVECs. By contrast, treatment of the HUVECs with 5 to 50 μM honokiol for 24 h before TNF-α stimulation resulted in a significant reduction of T98G cells adhering to ECs (Fig. 3).

Cancer cell invasion is important during the formation of distant metastases. Therefore, an in vitro invasion assay was performed to assess whether honokiol could inhibit glioblastoma invasion. T98G cells not treated with honokiol exhibited significant migration across a transwell membrane; by contrast, treatment with 5 to 50 μM honokiol significantly inhibited cancer cell invasion (Fig. 4).

Subsequently, we examined the cell viabilities of HUVECs and T98G cells in response to honokiol. When HUVECs and T98G cells were treated with varying doses of honokiol (1, 5, 10, 20 or 50 μM) for 24 h, honokiol significantly suppressed cell viability of T98G cells at doses of 10 μM or more; 50 μM of honokiol decreased cell viability of T98G cells by approximately 77% (Fig. 5A). Although honokiol also decreased the cell viability of HUVECs, honokiol-mediated cytotoxicity was not significant at doses lower than 20 μM, and the 50 μM dose was less toxic to HUVECs than to T98G cells (Fig. 5B).

Fig. 5A shows that honokiol significantly induced T98G cell death from 10 μM, a lower dose than was toxic to HUVECs. To confirm that honokiol-induced cytotoxicity was due to the induction of apoptotic cell death in T98G cells, we performed a TUNEL assay and also assayed the levels of the anti-apoptotic protein, Bcl-2, and the pro-apoptotic protein, Bax, by western blot analysis. Cells were treated with honokiol (toxic doses; 10, 20 or 50 μM) for 24 h, and TUNEL-positive cells were determined as described in Materials and methods. As shown in Fig. 6, honokiol effectively increased the number of TUNEL-positive cells at 10, 20 and 50 μM, suggesting that honokiol-induced cytotoxicity was due to the induction of apoptotic cell death. Moreover, western blot analysis showed that honokiol significantly increased pro-apoptotic Bax protein levels and decreased anti-apoptotic Bcl-2 levels in T98G cells at doses of 10 μM or more (Fig. 7), corresponding to the honokiol-induced cell death that also occurs at doses of 10 μM or more. These results suggest that honokiol induces apoptotic cell death in glioblastoma cells through the upregulation of the Bax/Bcl-2 ratio.