3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), 4,6-diamidino-2-phenylindole (DAPI), and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS), Roswell Park Memorial Institute (RPMI)-1640 medium, and penicillin-streptomycin were obtained from Gibco-BRL (Grand Island, NY, USA). Caspase activity assay kits were obtained from R&D Systems (Minneapolis, MN, USA). Extracellular signal-regulated kinase (ERK)-specific inhibitor, PD98059, c-Jun N-terminal kinase (JNK)-specific inhibitor, SP600125, p38 mitogen-activated protein kinase (MAPK)-specific inhibitor, SB203580, and Akt inhibitor, LY290042, were purchased from Calbiochem (San Diego, CA, USA). The DNA staining kit (Cycletest™ Plus Kit) and the enhanced chemiluminescence (ECL) kit were purchased from Becton-Dickinson (San Jose, CA, USA) and Amersham Pharmacia Biotech (Arlington Heights, IL, USA), respectively.

Antibodies specific for Fas, Fas ligand (FasL), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), Bcl-2, Bcl-xL, Bax, Bad, Bid, X-linked inhibitor of apoptosis protein (XIAP), inhibitor of apoptosis protein (IAP)-1, IAP-2, survivin, caspase-3, -8 and -9, poly(ADP-ribose) polymerases (PARP), β-catenin, and phospholipase Cγ1 (PLCγ1) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Antibodies specific for ERK, JNK, p38 MAPK, Akt, phospho (p)-ERK, p-JNK, p-38 MAPK and p-Akt were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Antibodies specific for death receptor (DR)4 and DR5 were obtained from Calbiochem. Anti-actin antibody was obtained from Sigma-Aldrich.

The stem bark of D. morbifera used in this research was obtained from Dongeui University Oriental Hospital (Busan, Korea). To prepare the ethanol extract of D. morbifera stem bark (EEDM), the dried stem was extracted twice with 70% ethanol (with 24 h reflux), and the extract was then concentrated under reduced pressure. The decoction was filtered, lyophilized, and stored at 4°C until use. The dried extract yield from the starting crude materials was ~17.8% (w/w). The lyophilized powder was dissolved in dimethyl sulfoxide (DMSO), filtered through a 0.22-μm syringe filter to create a stock solution, and then adjusted to final concentrations using complete RPMI-1640 medium.

U937 human leukemia cells, Chang liver cells, and WI-38 lung fibroblast cells were purchased from the American Type Culture Collection (Rockville, MD, USA) and maintained at 37°C in 95% humidified air and 5% CO₂ in RPMI-1640 medium that was supplemented with 10% FBS and 1% penicillin/streptomycin. Inhibition of cell proliferation was determined using an MTT assay. Briefly, cells were seeded in 6-well plates and treated with various concentrations of EEDM for 12 and 24 h. After treatment, the MTT working solution (0.5 mg/ml) was added to each well and incubated continuously at 37°C for 3 h. The culture supernatant was removed from the wells, and DMSO was added to completely dissolve the formazan crystals. The absorbance of each well was measured at a 540 nm wavelength with an ELISA reader (Molecular Devices, Sunnyvale, CA, USA). Morphological observations of cultured cells were carried out with an inverted microscope (Carl Zeiss, Germany).

For nuclear DAPI staining, the cells were harvested and washed with ice-cold phosphate-buffered saline (PBS) and fixed with 3.7% paraformaldehyde (Sigma-Aldrich) in PBS for 10 min at room temperature. The fixed cells were washed with PBS and stained with 2.5 μg/ml DAPI solution for 10 min at room temperature. The cells were then washed twice with PBS and analyzed with a fluorescence microscope (Carl Zeiss).

Following EEDM treatment, the cells were lysed in a buffer containing 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, and 0.5% Triton X-100 for 1 h at room temperature. The lysates were vortexed and cleared by centrifugation at 19,000 × g for 30 min at 4°C. A 25:24:1 (v/v/v) equal volume of neutral phenol:chloroform:isoamyl alcohol (Sigma-Aldrich) was used to extract DNA in the supernatant, followed by electrophoretic analysis on 1.5% agarose gels containing 0.1 μg/ml ethidium bromide (EtBr; Sigma-Aldrich).

The cells were collected, washed with cold PBS, and fixed in 75% ethanol at 4°C for 30 min. The DNA content of the cells was measured using a DNA staining kit according to the manufacturer’s instructions. Then flow cytometric analyses were carried out using a flow cytometer, and the relative DNA content was determined using CellQuest software, with the presence of red fluorescence as a marker. The MMP values were determined with the dual-emission potential-sensitive probe, JC-1. The cells were collected and incubated with 10 μM JC-1 for 20 min at 37°C in the dark. The cells were then washed once with PBS and analyzed with a flow cytometer (22).

Cells were lysed for 30 min with lysis buffer (20 mM sucrose, 1 mM EDTA, 20 μM Tris-Cl, pH 7.2, 1 mM DTT, 10 mM KCl, 1.5 mM MgCl₂, 5 μg/ml pepstatin A, 10 μg/ml leupeptin, and 2 μg/ml aprotinin) to prepare the total protein. The supernatants were collected, and the protein concentrations were determined using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). For western blot analysis, an equal amount of protein was subjected to electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH, USA) by electroblotting. Blots were probed with the desired antibodies for 1 h, incubated with the diluted enzyme-linked secondary antibodies, and visualized with the ECL solution according to the recommended procedure.

Caspase activity was determined with colorimetric assay kits, which use synthetic tetrapeptides [Asp-Glu-Val-Asp (DEAD) for caspase-3, Ile-Glu-Thr-Asp (IETD) for caspase-8, Leu-Glu-His-Asp (LEHD) for caspase-9] labeled with p-nitroaniline (pNA). Briefly, cells were lysed in the supplied lysis buffer according to the manufacturer’s protocol. The supernatants were collected and incubated with the supplied reaction buffer containing DTT and DEAD-pNA, IETD-pNA, or LEHD-pNA as the substrate at 37°C. The reactions were measured by changes in absorbance at 405 nm using a microplate reader.

All experiments are expressed as the means ± standard deviations (SDs) of at least 3 separate tests. The Student’s t-test was used for single-variable comparisons, and a p-value <0.05 was considered to indicate a statistically significant result.

To investigate whether EEDM inhibits U937 cell growth, the cells were treated with various concentrations (0–200 μg/ml) of EEDM for 12 and 24 h, and viability was measured with an MTT assay. Compared to the untreated control, after treatment with EEDM, as indicated in Fig. 1A, EEDM had a strong inhibitory effect on cell proliferation of U937 cells in a dose- and time-dependent manner. However, EEDM had no antiproliferative effect on Chang normal liver and WI-38 lung-derived cell lines. Direct observation using an inverted microscope showed that numerous morphological changes occurred in the U937 cells treated with EEDM. In particular, cell shrinkage and cytoplasmic condensation appeared in a concentration-dependent manner after EEDM treatment (Fig. 2A).

Further experiments were conducted to determine whether the inhibition of cell viability observed in the presence of EEDM was the result of apoptotic cell death in U937 cells. To examine the apoptosis morphologically, the nuclei of untreated and EEDM-treated cells were stained with DAPI solution and then observed. Following treatment of U937 cells with various concentrations of EEDM for 24 h, the chromatin stained with DAPI had a characteristic condensed and fragmented appearance in a concentration-dependent fashion (Fig. 2B). In addition, following agarose gel electrophoresis of the DNA from cells treated with EEDM, a characteristic ladder pattern of discontinuous DNA fragments was observed; however, DNA fragmentation was barely detected in the control cells (Fig. 2C). We next quantified the apoptotic dead cells using a flow cytometer. As shown in Fig. 2D, EEDM treatment resulted in a significant increase in the number of U937 cells in the apoptotic sub-G1 phase, and this response occurred in a concentration-dependent manner. These results confirmed that EEDM inhibits the proliferation of U937 cells by inducing apoptosis.

To investigate the mechanisms underlying EEDM-induced apoptosis, the expression level of several apoptotic factors was analyzed. Fig. 3A demonstrates that, after the cells were exposed to EEDM, the levels of the death receptor-related proteins Fas, FasL and TRAIL, but not DR4 and DR5, were concentration-dependently increased. Fig. 3A also shows that the levels of the anti-apoptotic Bcl-2 family of proteins (Bcl-2 and Bcl-xL) were decreased in response to EEDM; however, the levels of pro-apoptotic Bax and Bad remained unchanged. In addition, although we did not detect the truncated form of the pro-apoptotic protein Bid, EEDM decreased the total form of the Bid proteins, reflecting Bid cleavage and activation. Under these conditions, levels of the anti-apoptotic IAP family of proteins such as XIAP, cIAP-1 and survivin, which bind to caspases and lead to their inactivation, were inhibited by EEDM treatment in a concentration-dependent manner.

Mitochondrial membrane permeabilization, accompanied by the collapse of the electrochemical gradient across the mitochondrial membrane, is a key event during cellular apoptosis. To investigate whether mitochondrial dysfunction was involved in EEDM-induced U937 cell apoptosis, we used flow cytometric analysis with JC-1 staining to examine the change in the MMP values after EEDM treatment. JC-1 is a lipophilic and cationic dye that selectively enters mitochondria. In healthy cells with high mitochondrial potential, JC-1 forms J-aggregates with intense red fluorescence (590 nm), whereas under apoptotic conditions, the mitochondrial membrane potential collapses. Thus, JC-1 does not accumulate within the mitochondria but remains in the cytoplasm in a monomeric form showing green fluorescence (525 nm). As shown in Fig. 3B, JC-1 fluorescence shifted from a JC-1-red-bright/JC-1-green-bright signal in the control cells to a JC-1-green-bright/JC-1-red-dim signal in the EEDM-treated cells in a dose-dependent manner, indicating EEDM induced loss of the MMP in U937 cells.

To investigate the apoptotic cascade induced by EEDM, U937 cells were exposed to various concentrations of EEDM for 24 h, after which the expression level and activity of caspase-3, -8 and -9 were measured. As shown in Fig. 4, the expression of pro-caspase-3, -8 and -9 decreased following EEDM treatment, which was associated with a significant concentration-dependent increase in the activity of these caspases compared with the untreated control cells. Furthermore, subsequent western blot analysis revealed that EEDM induced proteolytic degradation of PARP, β-catenin, and PLCγ1, downstream target proteins of activated caspase-3. Cleavage fragments of these proteins gradually increased over time.

To confirm the role of caspase activation in EEDM-induced apoptosis, U937 cells were exposed to a broad-spectrum caspase inhibitor, z-VED-fmk, for 2 h before EEDM was added. As shown in Fig. 5, pretreatment with z-VED-fmk significantly attenuated EEDM-induced chromatin condensation, the formation of apoptotic bodies, and DNA fragmentation and restored the increased apoptosis. Furthermore, z-VED-fmk alone did not affect cell viability but reversed the anti-proliferative activity of EEDM (Fig. 5C). These data clearly indicate that EEDM-induced apoptosis in U937 cells was mediated by caspase activation.

To address whether the activation of the MAPK signaling pathway is involved in EEDM-induced apoptosis, we investigated whether members of the MAPK family of proteins are activated during EEDM-induced apoptosis. As Fig. 6A demonstrates, stimulation of U937 cells with EEDM led to rapid phosphorylation of MAPKs, including p38 MAPK, ERK and JNK, with peak levels of each phospho-MAPK observed 0.5 to 6 h after EEDM was added. However, the specific inhibitors of each protein (PD98059, ERK-specific inhibitor; SP600125, JNK-specific inhibitor and SB203580, p38 MAPK-specific inhibitor) did not block EEDM-induced cell death (Fig. 6B), indicating that EEDM-induced apoptosis is independent of the MAPK pathway. We next examined the involvement of the phosphatidylinositol-3-kinase (PI3K)/Akt pathway in EEDM-induced U937 apoptosis. As shown in Fig. 6A, the levels of phosphorylated Akt, a downstream kinase of PI3K, were time-dependently decreased in response to EEDM, while the total Akt protein levels remained constant during EEDM treatment. Similar to MAPK inhibitors, combination treatment with EEDM and LY294002, a specific inhibitor of PI3K, did not affect EEDM-induced apoptosis. These results suggest that EEDM-induced apoptosis is not associated with downregulation of the Akt signaling pathway.