Deltonin was obtained as previously described and its purity determined by high performance liquid chromatography (>98%) (19). The compound was prepared as a stock solution in DMSO [final concentration <0.05% (v/v)] and diluted in the relevant culture medium. ROS assay and JC-1 ΔΨm detection kits were obtained from Beyotime Institute of Biotechnology (Jiangsu, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), DMSO, N-acetylcysteine (NAC) and primary antibody against GAPDH were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against caspase-3 and -8, ERK1/2, phospho-ERK1/2 (Thr202/204), AKT and phospho-AKT (Ser473) were purchased from Cell Signaling Technology (Beverly, MA, USA). Primary antibodies against Bcl-2 (C-2), Bax (P19) and secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). All other chemicals and reagents were of the highest purity grade commercially available.

Human breast carcinoma cell line, MDA-MB-231, was obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in RPMI-1640 medium containing 10% fetal bovine serum (Gibco-BRL, Auckland, New Zealand), 100 U/ml penicillin and 100 μg/ml streptomycin in a humid chamber at 37°C under 5% CO₂(21).

The inhibitory effect of deltonin on cell viability was measured by MTT assay (22). Cells were seeded in 96-well plates (Costar Corning, Rochester, NY, USA) at a density of 3×10³ cells/well. Deltonin underwent serial dilution (0.5, 1.0, 2.0, 4.0, 8.0 μM) and DMSO (<0.05%) was used as a control. Absorbance at 570 nm was measured with Spectra Max M5 (Molecular Devices, LLC, Sunnyvale, CA, USA) following drug treatment (12, 24, 36, 48 h). All experiments were performed in triplicate. Dose- and time-dependent curves of deltonin-treated MDA-MB-231 cells were generated as the percentage cell growth inhibition, using the following formula: % inhibition = 1 - A570_{treated cells}/A570_{control cells} × 100. The 50% inhibiting concentration was calculated using SPSS software v13.0.

The use of Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) for flow cytometry provides a rapid and convenient assay for apoptosis as described previously (23). Cells were exposed to desired concentrations of deltonin for 24 h and the apoptotic index was assessed using an Annexin V-FITC apoptosis kit (KeyGEN, Nanjing, China) according to the manufacturer's instructions. For each analysis, 30,000 events were acquired on a forward and side scatter gate. Annexin V-positive PI-negative cells represented early apoptotic populations and Annexin V-positive PI-positive cells represented late apoptotic or secondary necrotic populations. Data acquisition and analysis were performed by fluorescence-activated cell sorting using list mode software (Becton-Dickinson, Franklin Lakes, NJ, USA).

Alterations in ΔΨm were analyzed by flow cytometry using a ΔΨm-sensitive molecular probe dye, JC-1, as described previously (24). The JC-1 dye bearing a delocalized positive charge enters the mitochondrial matrix due to the negative charge established by the intact ΔΨm. In healthy cells, JC-1 dye stains the mitochondria red due to formation of JC-1 aggregates. In apoptotic cells, JC-1 dye accumulates in the cytoplasm in monomeric form (green fluorescence) due to collapse of ΔΨm. Cells were treated with 3.0 μM deltonin for 0, 4, 8, 12, 16 and 24 h. Following this, cells were harvested, washed once in cold PBS and incubated with JC-1 dye at 37°C for 20 min. Stained cells were washed and re-suspended in 0.5 ml assay buffer and the fluorescence was measured using flow cytometry. The emission wavelengths of JC-1 monomers and JC-1 aggregates were 530 and 590 nm, respectively.

Intracellular ROS generation was monitored using an ROS assay kit and flow cytometry using fluorescence produced by 2′,7′-dichlorofluorescein following oxidation from 2′,7′-dichlorfluorescein-diacetate (DCFH-DA; Molecular Probes, Grand Island, NY, USA) (25). In brief, following treatment with 3.0 μM deltonin for 0, 4, 8, 12, 16 and 24 h, cells were harvested and then incubated with 10 μM DCFH-DA in a culture medium at 37°C for 30 min. Cells were washed and re-suspended in PBS. ROS generation was measured by flow cytometry.

To confirm the role of intracellular ROS in deltonin-induced apoptosis, a common quencher of ROS, NAC, was used to inhibit intracellular alteration of redox states (26). NAC was dissolved in PBS and adjusted to pH 7.4 to produce a 0.1 M stock solution. Cells were pre-incubated with 6 mM NAC for 2 h and then treated with 3 μM deltonin. After 8 h, alterations in ROS production were determined by flow cytometry. After 24 h, percentage of apoptotic cells and alterations in ΔΨm were analyzed by flow cytometry.

Western blot analysis was performed as described previously (27). Cells (1×10⁶/well) were grown in 6-well microplates overnight and treated with various concentrations (0, 1, 3, 5 μM) of deltonin for 24 h. Following this, cells were washed with ice-cold PBS and total cell lysates were prepared by RIPA buffer. Lysates was centrifuged at 12,000 g for 15 min at 4°C. The supernatant was collected and total protein concentrations were determined using the BCA assay, dissolved in 5X SDS sample buffer and denatured. Proteins were separated on 10-15% SDS-PAGE and transferred to polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA, USA). Membranes were incubated overnight at 4°C with the respective primary antibodies [caspase-3 and -8, poly (ADP ribose) polymerase (PARP), Bcl-2, Bax, ERK1/2, phospo-ERK1/2, AKT and phospo-AKT] and horseradish peroxidase-conjugated secondary antibodies at 37°C for 1 h. Reactive bands were identified using an enhanced chemiluminescent substrate to horseradish peroxidase (Amersham Pharmacia Biotech, Amersham, UK). Expression of GAPDH was used as a control.

Data are presented as mean ± SE and were analyzed for statistical significance using analysis of variance, followed by Scheffe's test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

The chemical structure of deltonin is presented in Fig. 1A. The cytotoxic effects of deltonin were investigated by MTT assay. As demonstrated in Fig. 1B, the maximum inhibition ratio obtained with 8 μM deltonin treatment was 89.40%. Deltonin inhibited the proliferation of MDA-MB-231 cells in a time- and concentration-dependent manner.

Flow cytometry analysis with an Annexin V-FITC apoptosis kit was also performed to identify the effects of deltonin. As shown in Fig. 2A, deltonin treatment induced apoptosis in MDA-MB-231 cells in a concentration-dependent manner. Compared with untreated cells, cells treated with 5 μM deltonin resulted in up to 75.7% apoptosis incidence, indicating that deltonin causes death in MDA-MB-231 cells by the induction of apoptosis.

Apoptosis is executed by the coordinated actions of caspase family members and is tightly regulated by Bcl-2 protein family members. Therefore, protein expression levels of these molecules were analyzed by western blot analysis. As demonstrated in Fig. 2B, treatment of MDA-MB-231 cells with deltonin led to activation of caspase-3 and -8. It is well known that activation of caspase-3 during apoptosis causes the cleavage of PARP, a major apoptotic enzyme. Deltonin increased levels of cleaved PARP in a dose-dependent manner, consistent with deltonin-induced apoptosis in MDA-MB-231 cells. Following this, the protein expression levels of Bcl-2 family members, Bcl-2 and Bax were determined. Fig. 2B shows a decrease in Bcl-2 and increase in Bax levels.

Change of ΔΨm using JC-1 was performed to investigate whether mitochondria are involved in deltonin-induced apoptosis. Representative flow cytometric results are presented in Fig. 3. Deltonin treatment led to a time-dependent increase in the number of green fluorescence-positive cells from 9.5 in untreated cells to 17.1, 23.3, 40.9, 49.3 and 58.6%.

ΔΨm loss is an early event in apoptosis induction, which is often accompanied by ROS production. Intracellular ROS was examined using DCFH-DA to analyze the role of ROS in deltonin-induced apoptosis. As demonstrated in Fig. 4, treatment of MDA-MB-231 cells with 3 μM deltonin for 0, 4, 8, 12, 16 and 24 h resulted in ROS increase from 10.3 to 66% from 0 to 8 h, however, ROS levels decreased to 13.7% at 24 h. These results revealed that the ROS burst is generated in a time-dependent manner.

To determine the association between intracellular ROS and deltonin-induced apoptosis, MDA-MB-231 cells were pretreated with 6 mM NAC for 2 h and then incubated with 3 μM deltonin. As revealed in Fig. 5A, pretreatment with NAC reduced the number of apoptotic cells, whereby the percentage of apoptotic cells decreased from 32.9 to 23.4%.

NAC markedly inhibited deltonin-induced accumulation of ROS, the intensity of DCFH-DA fluorescence decreased from 54.8 to 18.0% (Fig. 5B).

NAC is known to exert protective effects on cells, therefore, the ability of NAC to prevent depolarization of ΔΨm in deltonin-treated cells was invetigated. Fig. 5C reveals flow cytometry analysis of ΔΨm in the presence of absence of NAC (6 mM) pretreament. The results demonstrate that deltonin-induced apoptosis in MDA-MB-231 cells may be mediated by the mitochondrial pathway and, at least in part, by accumulation of ROS.

ERK and AKT are two notable pathways associated with tumor development. Inhibition of the ERK pathway or AKT activation has been identified to induce apoptosis in tumor cells (28-30). To investigate the involvement of ERK/AKT signal pathways in deltonin-induced apoptosis of MDA-MB-231 cells, levels of phosphorylated and unphosphorylated AKT and ERK1/2 were determined by western blot analysis. As shown in Fig. 6, treatment of deltonin decreased levels of phospo-ERK1/2 and -AKT in a dose-dependent manner compared with GAPDH (loading control). Results indicate that interference with ERK and AKT signal pathways may contribute to the anticancer activity of deltonin.