The human breast cancer cell line MDA-MB-468 was purchased from the Cell Bank of the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China) and stored in liquid nitrogen. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) culture medium containing 10% fetal bovine serum (FBS; both from Gibco, USA), 100 U/ml penicillin G, 2.5 μg/ml amphotericin B and 100 μg/ml streptomycin (complete medium) at 37°C with 5% CO₂ in a humidified atmosphere.

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Propidium iodide (PI)/RNase staining kit and Annexin V-FITC/7AAD kit were purchased from BD Pharmingen (San Diego, CA, USA). Antibodies against caspase-3 (#9662), cleaved caspase-3 (Asp175, #9664), cdc2 (#9116), phospho-Akt (Ser473/Thr308, #9275), Akt (pan, #4691), phospho-cdc2 (Tyr15, #4539), PARP (#9532), cyclin B1 (#12231), Bcl-2 (#2870), Bcl-xL (#2764), caspase-8 (#4790), caspase-9 (#9508), cleaved caspase-9 (Asp330, #9501), β-tubulin (#2128), and horseradish peroxidase-conjugated secondary antibodies were purchased from Cell Signaling Technologies (Beverly, MA, USA). Bad (N-term, 1541-1) was purchased from Abcam (Cambridge, UK).

The viability of cells treated with or without EO was measured by MTT assay. The MDA-MB-468 cells were placed into 96-well plates at a final concentration of 5×10³ cells/well in complete medium, and allowed to attach for 24 h. Subsequently the cells were treated with a range of concentrations of EO for 24, 48 and 72 h, then 20 μl MTT solution (5 mg/ml) was added, and the cells were incubated for another 4 h at 37°C in the dark. Formed formazan crystals were dissolved in 100 μl DMSO and the absorbance was measured at 570 nm on a microplate reader (Bio-Rad, USA). The IC₅₀ value was calculated by GraphPad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA, USA).

Cells were plated in 6-well plates (3×10⁵ cells/well) and treated with varying concentrations of EO for 72 h. For cell cycle analysis, cells were harvested, washed twice with ice-cold PBS and fixed in 70% ethanol at 4°C overnight, and stained with PI/RNase (0.5 ml/test, 1×10⁶ cells) for 15 min at room temperature before analysis. To quantify the apoptotic cells, the treated cells were washed twice with ice-cold PBS and stained with Annexin-V-FITC/7AAD according to the manufacturer's instructions. Samples were subsequently analyzed by flow cytometer (Guava Technologies; Merck KGaA, Darmstadt, Germany) and DNA content was quantified using ModFit software.

The MDA-MB-468 cells (3×10⁵) were seeded in 6-well plates for 24 h before the experiment. After the treatment with 2, 4, and 8 μM EO for 24 h, cells were harvested, washed twice with ice-cold PBS, and incubated with JC-1 (10 μg/ml) in the dark for 15 min at 37°C. Cells were washed three times with ice-cold PBS and analyzed by flow cytometry using emission wavelengths of 590 nm and 529 nm.

Cells were harvested, washed twice with ice-cold PBS after treatment with EO, lysed by incubation in RIPA buffer containing a protease inhibitors cocktail (1 mM phenylmethanesulfonyl fluoride and 1 μg/ml leupeptin) and a phosphatase inhibitors cocktail (1 mM sodium fluoride and 1 mM sodium orthovanadate) for 30 min on ice, and then centrifuged at 12,000 rpm for 15 min at 4°C. Supernatants were collected, and equal amounts of denatured proteins (heated samples at 100°C for 10 min) were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA), blocked with 5% nonfat milk at room temperature for 1 h, and incubated with the respective specific primary antibodies overnight at 4°C. The membranes were washed three times with Tris-buffered saline-5% Tween-20 (TBST) solution and incubated with a horseradish peroxidase-conjugated secondary antibody at room temperature for 2 h. Chemiluminescent detection was performed by ECL (Bio-Rad, USA).

All data are expressed as the mean ± SD of three independent experiments. Statistical significance was analyzed using a Student's t-test. The criterion of statistical significance was ^**p<0.01; ^***p<0.001.

To evaluate the anti-proliferative effect of EO in MDA-MB-468 human breast cancer cells, the cells were treated with indicated concentrations of EO for 24, 48 and 72 h. The cytotoxic effect was measured by MTT assay. As shown in Fig. 1B, EO induced cytotoxicity in the MDA-MB-468 cells in a concentration- and time-dependent manner. IC₅₀ at 72 h was 1.04 μM. The effects of EO on the MDA-MB-468 cells may be the result of the induction of apoptosis and/or inhibition of growth. Therefore, the authors investigated whether EO induces apoptosis in human breast cancer cells.

To further determine whether the growth-inhibitory effect of EO was related to the induction of apoptosis, treated cells were analyzed using Annexin V-FITC/7AAD staining by flow cytometric analysis. As shown in Fig. 2, after incubation with EO, the percentage of apoptotic cells was significantly increased. These results indicated that the induction of apoptotic cell death can be a potential mechanism of the anticancer effect of EO against human breast cancer cells.

The Δψm is an early event preceding caspase activation, and is regarded as a hallmark of apoptosis (18). Induction of apoptosis via mitochondrial pathways results in the loss of mitochondrial membrane potential. Therefore, we measured Δψm in EO-treated MDA-MB-468 cells using the membrane-permeable JC-1 dye. In apoptotic cells with low Δψm, JC-1 remains in the monomeric form, which has green fluorescence (19). As shown in Fig. 3, a marked increase in green fluorescence could be seen in the MDA-MB-468 cells treated with 2, 4 and 8 μM of EO. These results demonstrated that EO induced Δψm disruption in breast cancer cells.

As known the activation of caspases plays important roles in cancer cell death. To confirm whether caspases are involved in EO-induced MDA-MB-468 cell death, we next examined the activation of caspases and the expression of poly(ADP-ribose) polymerase (PARP) by western blot analysis. As shown in Fig. 4, EO treatment significantly decreased the protein levels of pro-caspase-3, pro-caspase-8 and pro-caspase-9, while increasing the protein levels of cleaved caspase-3 and caspase-9. To further investigate the enzymatic activation of caspase-3, we measured cleaved PARP which is a substrate of caspase-3 and an enzyme that protects DNA. The formation of the fragment of PARP was detected in cells treated with EO (Fig. 4). These results demonstrated that EO induced apoptosis in the MDA-MB-468 cells by the activation of caspases.

To further investigate the role of caspase activation in EO-induced apoptosis, the effect of the pan-caspase inhibitor Z-VAD-FMK in preventing EO-induced cell death was examined. As shown in Fig. 5A, when cells were treated with 8 μM of EO, the percentage of apoptotic cells reached 65.01% at 24 h. However, when cells were pre-treated with caspase inhibitor Z-VAD-FMK for 2 h, the percentage of apoptotic cells was reduced to 22.44% after 24 h. Moreover, western blot analysis showed that cleavage of caspase-3 and PARP were inhibited (Fig. 5B). These results indicated that EO induced apoptosis mainly by caspase-dependent mechanisms in the MDA-MB-468 cells.

To investigate whether EO modulates the cell cycle of the MDA-MB-468 cells, the cells were treated with the indicated concentrations (0, 2, 4 and 8 μM) of EO for 24 h. The detection of cell cycle distribution was performed by flow cytometry. As shown in Fig. 6A, the G2/M phase distribution was increased from 12.67% in the vehicle control group to 31.60% in a concentration-dependent manner after 8 μM of EO treatment. To elucidate the molecular mechanism, we next detected the expression of key molecules (cyclin B1, cdc2 and p-cdc2) which regulate the G2/M phase transition in the EO-treated MDA-MB-468 cells. As shown in Fig. 6B, treatment with EO decreased the protein levels of cyclin B1 and cdc2 while increasing the protein level of p-cdc2 (Tyr15) in a dose-dependent manner. These results suggested that the cell cycle arrest in G2/M phase may be associated with the anticancer effect of EO on human breast cancer cells.

The Bcl-2 family of proteins, such as Bcl-2 and Bax, are upstream signals of caspase activation and play important roles in regulating mitochondrial-related apoptosis (20). The Bcl-2 family includes anti-apoptotic and pro-apoptotic proteins. The release of proteins from the inner-membrane space of mitochondria is one of the central events in the apoptotic process, which can lead to the activation of caspases and the ultimate demise of the cell (21). To elucidate further the anticancer mechanism, the role of the Bcl-2 family of proteins in EO-induced apoptosis was investigated. As shown in Fig. 7, after treatment with EO, the expression of anti-apoptotic Bcl-xl and Bcl-2 was significantly decreased, and the expression of pro-apoptotic Bax and Bad was increased. Collectively, these results suggest that EO may induce apoptosis of the MDA-MB-468 cells through Bcl-2 degradation.

To further explore the possible mechanism of EO on the induction of apoptosis in breast cancer cells, we assessed its effect on the Akt signaling pathways. The Akt signaling pathways have been shown to be critical regulators of cell growth, differentiation and survival (22,23). Sesquiterpene lactones can promote cancer cell survival by activating the PI3K/Akt pathway (24). The Akt pathway is a major anti-apoptotic signaling pathway, and previous studies have shown that chemotherapy drugs induce apoptosis of tumor cells by inhibiting the Akt pathway (25–27). Thus, we determined the protein expression of Akt in the MDA-MB-468 cells and found that EO significantly reduced the expression of both Akt and p-Akt which is an active form of Akt in a dose-dependent manner (Fig. 7). These data suggest that the Akt pathway may be an effective target for EO in the MDA-MB-468 cells.