The MDA-MB-231 breast cancer line (American Type Culture Collection, Manassas, VA, USA) was cultured in the laboratory of the Department of Pharmacy, Bengbu Medical College (Bengbu, China). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin, and were maintained at 37°C with 5% CO₂ in a humidified atmosphere. All the experiments were performed on logarithmically growing cells.

DMEM, FBS and phosphate-buffered saline (PBS) were purchased from Thermo Fisher Scientific, Inc. (Grand Island, NY, USA). MTT [also known as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was obtained from Sigma-Aldrich (Castle Hill, Australia). Penicillin and streptomycin were purchased from North China Pharmaceutical Group Corp., (Shijiazhuang, China). Propidium iodide (PI)/Annexin V-FITC was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO) was obtained from Amresco (Solon, OH, USA). Mouse anti-human β-actin (1:10,000; 66009-1-Ig) polyclonal antibody and rabbit anti-B-cell lymphoma-2 (Bcl-2; 1:1,000; 12789-1-AP) and rabbit anti-Bcl-2-associated X protein (Bax; 1:1,000; 23931-1-AP) polyclonal rabbit anti-human antibodies were purchased from Proteintech Group, Inc. (Rosemont, IL, USA). The mitochondrial membrane potential (ΔΨm) assay kit (C2006), JC-1, bicinchoninic acid protein concentration kit (P0011) and crystal violet stain were purchased from the Beyotime Institute of Biotechnology (Shanghai, China).

The effect of the P. odoratum extract on cell viability was determined using an MTT assay. P. odoratum extract was socked in MeOH for 3 days isolate the filtrate, MeOH extract was obtained using rotary evaporators. MeOH extract was dissolved in a isopyknic mixture of EtOAc and H₂O to obtained the EtOAc layer and the EtOAc extract was isolated using rotary evaporators. Subsequently, EtOAc extract was dissolved in a isopyknic mixture of Hexane and H₂O to obtain the Hexane layer, and P. odoratum extract was subsequently obtained using rotary evaporators. Briefly, the MDA-MB-231 cells were seeded at a density of 1×10⁴ cells/well in 96-well plates with 100 ml growth medium. The cells were randomly divided into three groups as follows: Control (no treatment), P. odoratum extract (treatment with extract alone) and blank control (no cells, no treatment, just growth medium) groups. Cells in the P. odoratum extract group were then incubated for 24 h in the presence of 0, 0.001, 0.01 or 0.1 mg/ml P. odoratum extract. Next, 15 µl MTT (5 mg/ml) was added to the cells, which were incubated in the dark at 37°C for 4 h. Subsequent to removal of the MTT solution, the formazan product (crystals) was dissolved in 150 µl DMSO by shaking the plates for 10 min. Optical density (OD) was then determined at 490 nm using a Synergy HT multi-detection microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). The experiments were performed in triplicate, and three parallel samples were measured each time. For calculation of the cell proliferation rate (%), the following formula was used: OD (experimental group) / OD (control group) × 100.

MDA-MB-231 cells in the logarithmic growth period, were seeded at a density of 1×10⁴ cells/well in 6-well plates with 2 ml growth medium and incubated for 24 h. Next, the cells were treated with different concentrations of the P. odoratum extract (0, 0.02, 0.04 and 0.06 mg/ml) for 5 days and maintained at 37°C in 5% CO₂ in a humidified atmosphere. The cells were then washed twice with pre-chilled PBS, fixed with paraformaldehyde for 10 min and stained with 2% crystal violet for 10 min. Double-distilled water was used to clean the cells until the color of the sample turned transparent, and the cells were then dried at room temperature. Colonies contained 50–150 cells. Colony numbers were counted under a light microscope at a magnification of ×40.

A single cell suspension was prepared from MDA-MB-231 cells in the logarithmic growth period and counted using flow cytometry (Accuri C6; BD Biosciences, Franklin Lakes, NJ, USA). Annexin-V FITC/PI staining was performed according to the manufacturer's instructions to assess cellular apoptosis. Cells were seeded in 6-well culture plates (1×10⁴ cells/well) for 24 h prior to the cell apoptosis assay. Next, the cells were incubated with the P. odoratum extract at increasing concentrations (0, 0.02, 0.04 and 0.06 mg/ml) for 24 h and then washed twice with pre-chilled PBS. The cells were resuspended in 500 µl binding buffer, following centrifugation at 13,223 × g for 10 min. Subsequently, 100 µl cell suspension was transferred to 5-ml Eppendorf tubes, and 5 µl PI/Annexin V-FITC solution was added to each tube and mixed for 15 min (4°C; dark). Following incubation for 15 min at 20°C, the apoptosis rate was determined using an BD Accuri C6 flow cytometer, and the results were analyzed using the flow cytometer software.

Stable expression cells were washed three times with cold PBS (1 ml/well) and digested with trypsin in lysis buffer for 30 min on ice. The protein concentrations of the cell lysates were detected using a bicinchoninic acid protein concentration kit. The protein sample was mixed with the sample buffer (2:1) and boiled at 100°C with the denaturation buffer for 5 min, and steps were taken to prevent protein degradation. Protein samples from each test group (30 µg) were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A condensation protein electrophoresis gel was run at 70 V for 30 min and 150 V for 90 min, and the proteins were transferred onto polyvinylidene fluoride membranes at 50 V in an ice bath for 90 min. The membrane was then blocked in 5% skim milk, followed by an overnight incubation at 4°C with the primary antibodies against β-actin. Bcl-2 and Bax (1:1000). Subsequently, the membrane was washed three times with Tris PBS (TPBS) and once with PBS, for 10 min each time. The sample was then labeled with the peroxidase-conjugated rabbit (109525) or mouse (117228) secondary antibodies (1:5,000; ZSGB Bio, Beijing, China) for 2 h at room temperature on a shaker. Next, the membrane was washed with TPBS, and the bands were visualized using western lightning with enhanced chemiluminescence (EMD Millipore, Billerica, MA, USA). β-actin was used as the protein control, and the gray values of the protein bands in each group were analyzed using Quantity One 4.6.8.27 gel image analysis software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The experiments were repeated at least twice to confirm the results.

The mitochondrial stability was assessed using a ΔΨm assay kit with JC-1. MDA-MB-231 cells in the logarithmic growth period were seeded at a density of 2×10⁵ cells/well in 12-well plates with 1 ml growth medium and incubated for 24 h. Next, the cells were treated with 0.1 mg/ml of the drug. After treatment for 24 h, the cells were washed once with PBS and then incubated with 1 ml JC-1 fluorescent dye for 20 min in the dark at 37°C. Subsequently, the cells were washed twice with pre-chilled buffer solution. The ΔΨm was imaged using a fluorescence microscope (Olympus Corp., Tokyo, Japan) at 550 nm excitation and 570 nm emission.

All experiments were performed at least in triplicate. Data are presented as the mean ± standard error of the mean. A double-sided Dunnett's test was used for between-group comparisons. All statistical analyses were performed using the SPSS version 13.0 software (SPSS, Inc., Chicago, IL, USA) and statistically significant differences were indicated by P<0.05.

An MTT assay was performed to determine the effect of P. odoratum extract on the proliferation of MDA-MB-231 cells. As shown in Fig. 1, the viability of MDA-MB-231 cells was altered following 24-h treatment with various concentrations of the P. odoratum extract up to 0.1 mg/ml. The extract inhibited MDA-MB-231 cell proliferation in a dose-dependent manner (P<0.05).

Furthermore, the colony forming ability of the MDA-MB-231 cells was analyzed using a colony formation assay. As shown in Fig. 2 (P<0.05), a significant inhibitory effect was observed in cells treated with increasing concentrations of the P. odoratum extract (0, 0.02, 0.04 and 0.06 mg/ml) for 5 days, while low concentration significantly inhibited the cell colony formation. These results suggest that the P. odoratum extract has inhibitory effects on breast cancer cell proliferation.

To determine whether the reduced cell viability was due to apoptosis, PI/Annexin V FITC double staining using the Accuri C6 flow cytometry was conducted. As indicated in Fig. 3 (P<0.05), MDA-MB-231 cells treated with increasing concentrations (0.02, 0.04 and 0.06 mg/ml) of the extract showed an increase in the ratio of apoptotic cells (7.9, 18.1, and 33.2%, respectively) for 24 h. These data showed that the P. odoratum extract induced dose-dependent apoptosis in the cells.

MDA-MB-231 cells were treated with the 0.1 mg/ml P. odoratum extract for 0, 6, 16, and 24 h, and western blot analysis was used to investigate the effect on the protein expression levels of Bcl-2 and Bax. The results showed that, compared with the control group, there was a time-dependent decrease and increase in the protein expression levels of Bcl-2 and Bax, respectively (Fig. 4; P<0.05). These findings indicate that the extract treatment resulted in downregulation of Bcl-2 and upregulation of Bax. β-actin expression was used as an internal control, and its level was not changed.

The ΔΨm following JC-1 staining was also examined using fluorescence analysis. The results indicated that there was a significant change in the ΔΨm loss in MDA-MB-231 cells following treatment with the P. odoratum extract (0.1 mg/ml, 24 h). As shown in Fig. 5, fluorescence conversion from red to green was observed in response to the extract treatment. This illustrates the early apoptosis that occurred in the cells subsequent to treatment. The intensity of the JC-1 staining was measured using fluorescence microscopy. JC-1 passed across the membrane into the living cells and aggregated in the mitochondrial membrane, and the concentration of JC-1 increased as mitochondrial membrane potential increased. When the ΔΨm is high, JC-1 accumulates in the mitochondrial matrix forming J aggregates and red fluorescence is produced. By contrast, when the ΔΨm is low, JC-1 cannot accumulate in the mitochondrial matrix and the monomer with the green fluorescence is produced. Therefore, the ΔΨm can be easily determined based on the fluorescent color change, and a decrease in ΔΨm is a sign of early apoptosis. Change in fluorescence from red to green induced by JC-1 can easily be detected with the decline of the membrane potential. Therefore, JC-1 staining can be used as an index for the detection of early apoptosis.