All the chemicals were purchased from Sigma-Aldrich (Beijing, China) unless otherwise stated. Evodiamine was purchased from Tauto Biotech. Co., Ltd. (Shanghai, China) and purity (>99%) was determined by HPLC, conducted at the Central Research Lab of Bethune Second Hospital of Jilin University (Changchun, China) as described in previous studies (15,19). The chemical structure of evodiamine is shown in Fig. 1A. The apoptosis assay kit was purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). Antibodies specific to caspase-8, cleaved caspase-3 and β-actin were purchased from Beyotime Institute of Technology (Shanghai, China), whereas the antibody targeting death receptor (DR)5 was purchased from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit and goat anti-mouse) were purchased from Sigma-Aldrich. The enzyme-linked immunosorbent assay (ELISA) kit for human DR4 was purchased from ROTRN Shanghai Biotechnology Co., Ltd. (Shanghai, China). TRAIL was purchased from Abcam Inc. (Cambridge, MA, USA).

U87 glioblastoma cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum in 5% CO₂ at 37°C. Cells were treated with evodiamine dissolved in dimethyl sulfoxide (DMSO), at a final DMSO concentration of 0.5%, or with DMSO alone for 24 h. DMSO-treated cells were used as the control.

Cell viability was determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay as previously described (26). Briefly, U87 glioblastoma cells were treated with various concentrations of evodiamine (0–20 μM) or TRAIL (0–50 ng/ml) for 24 h. Following treatment, the MTT reagent was added (500 μg/ml) and cells were further incubated at 37°C for 4 h. Subsequently, 150 μl of DMSO were added to dissolve the formazan crystals, and the absorbance was measured at 570 nm (A₅₇₀) in a Multiskan™ GO Microplate spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The percentage of cell viability was calculated as follows: Cell viability (%) = (A₅₇₀sample − A₅₇₀blank)/(A₅₇₀control − A₅₇₀blank) × 100.

Living and dead cells were quantified using the fluorescent probes calcein acetoxymethylester (AM) and propidium iodide (PI) as previously described (1,27). Calcein AM is highly lipophilic and cell membrane-permeable. In viable cells, it is converted into calcein by esterases and emits strong green fluorescence. Thus, it stains only viable cells. PI, a dye used for nuclear staining, is cell membrane-impermeable. Cells with impaired plasma membrane integrity are stained red due to the entry of PI and failure to retain calcein. Since both calcein and PI are excited at 490 nm, simultaneous monitoring of viable and dead cells is feasible under a fluorescence microscope (1X71; Olympus Corporation, Tokyo, Japan). To quantify living and dead cells, the cells were treated with evodiamine (10 μM) and TRAIL (50 ng/ml) either separately or in combination (evodiamine 10 μM + TRAIL 50 ng/ml) for 24 h. Following incubation, cells were collected, washed with phosphate-buffered saline (PBS), and incubated with PBS containing 2 μM calcein AM and 4 μM PI, for 20 min in the dark, at room temperature. A total of 100 cells were then counted under the fluorescence microscope in order to quantify the viable and dead cells.

U87 glioblastoma cells were treated with evodiamine and TRAIL either separately or in combination, for 24 h, as described in the Live/Dead assay. Following treatment, cells were harvested, washed with PBS and resuspended in 500 μl of binding buffer, containing 5 μl Annexin V-fluorescein isothiocyanate (FITC) and 5 μl PI. The cells were left in the dark for 15 min according to the kit instructions (KeyGen). The percentage of apoptotic cells was evaluated on a Epics XL flow cytometer (Beckman Coulter, Brea, CA, USA).

The level of DR4 in U87 glioblastoma cells was measured spectrophotometrically using the human DR4 ELISA kit. Briefly, U87 cells were treated with 10 μM evodiamine for 24 h. Following treatment, cells were collected by centrifugation (700 × g), and cell lysates were prepared according to the kit instructions. The level of DR4 in the cell lysates was quantified spectrophotometrically at 450 nm on a Synergy H1 Hybrid Microplate Reader (H1MD; Bio-Tek, Beijing, China).

U87 glioblastoma cells were treated with evodiamine (10 μM) and TRAIL (50 ng/ml) either separately or in combination (evodiamine 10 μM + TRAIL 50 ng/ml), for 24 h. Following treatment, cells were collected by centrifugation, and proteins were isolated as previously described (27,28). The protein concentrations were determined on a NanoDrop 1000 (Thermo Fisher Scientific Inc.) spectrophotometer. A total of 30 μg of protein was subjected to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a polyvinylidine difluoride membrane. After blocking with 5% (w/v) non-fat milk and washing with Tris-buffered saline with Tween-20 (TBST), the membrane was incubated for 2 h at room temperature with antibodies targeting DR5 (dilution, 1:1,000), cleaved caspase-3 (1:1,000), caspase-8 (1:1,000) and β-actin (1:400). After washing, the blot was incubated with horseradish peroxidase-conjugated goat anti-rabbit or -mouse IgG secondary antibodies (1:5,000) for 1 h at room temperature. Following a TBST wash, the signals were detected using a ChemiLucen™ Plus kit (EMD Millipore, Billerica, MA, USA).

Results were expressed as mean ± standard error of the mean (SEM). Comparisons between two groups were performed with Student’s t-tests, and between multiple groups with a one-way analysis of variance (ANOVA), followed by the Tukey’s Multiple Comparison test. P<0.05 was considered to indicate statistically significant differences.

First, we measured the inhibitory effect of evodiamine and TRAIL on growth of U87 glioblastoma cells using the MTT assay. Evodiamine significantly inhibited the growth of U87 cells in a dose-dependent manner, as shown in Fig. 1B. The half maximal inhibitory concentration (IC₅₀) value of evodiamine against U87 glioblastoma cells was 12 μM. However, TRAIL (1–50 ng/ml) alone did not inhibit the growth of U87 glioblastoma cells within 24 h. These results were further confirmed by the Live/Dead assay, as shown in Fig. 2. Next, we investigated whether evodiamine can sensitize U87 glioblastoma cells to TRAIL. For this purpose, we treated the cells with 10 μM evodiamine and 50 ng/ml TRAIL for 24 h. The combined treatment significantly increased cell death in U87 glioblastoma cells as compared to evodiamine treatment alone (Fig. 2). The above-mentioned concentrations of evodiamine and TRAIL were employed in all cotreatment experiments performed in this study.

To confirm that evodiamine + TRAIL-induced cell death occurs via apoptosis, we performed flow cytometry analysis. Cells were treated with evodiamine (10 μM) and TRAIL (50 ng/ml) either separately or in combination for 24 h, and the percentages of cells undergoing apoptosis/necrosis were determined after staining with Annexin V-FITC and PI. As expected, TRAIL alone did not induce apoptosis, while evodiamine significantly induced apoptosis in U87 glioblastoma cells after 24 h (Fig. 3). Combined treatment with evodiamine and TRAIL significantly enhanced apoptosis compared to treatment with each drug alone. The data demonstrated that evodiamine sensitizes U87 glioblastoma cells to TRAIL-induced apoptosis.

Since U87 glioblastoma cells are resistant to TRAIL-mediated apoptosis, a number of chemotherapeutic agents have been investigated for their ability to sensitize tumor cells to TRAIL-mediated apoptosis by inducing the expression of death receptors. Therefore, we examined here whether evodiamine can induce the expression of DR4 and DR5 in U87 glioblastoma cells. We measured the expression of DR4 and DR5 in U87 glioblastoma cells before and after treatment with evodiamine, using ELISA and western blot analysis, respectively. As shown in Fig. 4, evodiamine treatment increased the expression of both DR4 and DR5 as compared to the control.

To further evaluate the activation of downstream signaling related to apoptosis following the increased expression of DR4 and DR5, we measured the expression of caspase-8 and caspase-3. As shown in Fig. 5, TRAIL treatment alone did not affect the level of caspase-8 and cleaved (activated) caspase-3; however, evodiamine induced the expression of caspase-8 and cleaved caspase-3. Combined treatment with evodiamine and TRAIL further increased the expression of caspase-8 and cleaved caspase-3. These data suggested that the apoptotic effect of evodiamine + TRAIL may be due to caspase activation.