XHP was purchased from Tong Ren Tang Technologies Co., Ltd. (Beijing, China). Dimethyl sulfoxide (DMSO) and 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Caspase-3 monoclonal antibody (cat. no. ab32351; 1:3,000 dilution) was purchased from Abcam (Cambridge, UK). Cyclin dependent kinase 2 (CDK2) (cat. no. 2546; 1:5,000 dilution), cyclin A (cat. no. 4656; 1:5,000 dilution), cyclin E (cat. no. 4129; 1:5,000 dilution), p21^Cip1 (cat. no. 2947; 1:5,000 dilution), caspase-8 (cat. no. 9746; 1:4,000 dilution), B-cell lymphoma 2 (Bcl-2; cat. no. 2870; 1:5,000 dilution) and Bcl-2-associated X protein (Bax; ca. no. 5023; 1:5,000 dilution) monoclonal antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). β-actin rabbit monoclonal antibody (cat. no. TDY051; 1:5,000 dilution) was purchased from TDYbio (Beijing, China). Peroxidase-conjugated goat anti-rabbit immunoglobulin (Ig) G (cat. no. ZB-2306) and goat anti-mouse IgG (cat. no. ZF-0312; both 1:5,000 dilution) were purchased from the Zhongshang Goldenbridge-BIO (Beijing, China).

XHP (3 g) was immersed in 15 ml cold distilled water and stirred for 2 h at 4°C. Subsequently, XHP was extracted for 2 h at 37°C in an ultrasound oscillator and centrifuged at 1,500 × g for 5 min at 4°C. The supernatant was centrifuged again at 4,800 × g for 15 min at 4°C, and the final supernatant was filtered through a sterile microporous membrane (0.45 µm) and stored at −20°C. For treatment of the cells, AEXHP was diluted with Dulbecco's modified Eagle's medium (DMEM) to the desired concentrations.

The Hs578T human TNBC cell line was obtained from the cell bank of Chinese Academy of Medical Sciences (Shanghai, China). The cells were cultured in DMEM (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (Hyclone, Logan, UT, USA) and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin).

The MCF-10A human breast epithelial cell line also obtained from the cell bank of Chinese Academy of Medical Sciences. The cells were grown, maintained and treated in medium containing DMEM/F-12 (Gibco) 1:1 mix, supplemented with human insulin (10 µg/ml), epidermal growth factor (20 ng/ml), cholera toxin (100 ng/ml), hydrocortisone (0.5 µg/ml), 5% horse serum (Hyclone) and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin).

The cell lines were incubated in a humidified atmosphere containing 5% CO₂ at 37°C.

Cells (7.5×10³ or 1×10⁴/well) were seeded in 96-well plates (Corning Inc., Corning, NY, USA) and treated on the following day with various concentrations of AEXHP (0, 4, 8, 12 or 16 mg/ml) for 12, 24 or 48 h [the units ‘weight per volume’ refer to the quantity of solid XHP and the volume of water used for extraction, which was performed according to a previously published procedure (8)]. Subsequently, the cells were subjected to an MTT assay (9) and the optical density (OD) was determined at 570 nm using a 96-well microplate reader (Applied Biosystems; Thermo Fisher Scientific, Inc.). Since reduction of MTT only occurred in metabolically active cells, the OD was a measure of the viability of the cells. The viability rate was calculated according to the following formula: OD_treatment/OD_control × 100%.

Hs578T Cells (2.4×10⁵) were seeded in a 30-mm culture dish (Corning Inc.). On the following day, cells were treated with AEXHP (0, 4, 8 or 12 mg/ml). After 24 h of incubation, cells were trypsinized, washed with cold phosphate-buffered saline (PBS) and stained using an annexin V/FITC kit (BestBio, Shanghai, China). The cells were then analyzed on a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

Δψ_m loss was detected using a mitochondrial membrane potential assay kit (Beyotime Institute of Biotechnology, Haimen, China). In brief, Hs578T cells (2.4×10⁵) were seeded into a 30-mm culture dish. On the following day, the cells were treated with AEXHP (0, 4, 8 or 12 mg/ml). After 24 h of incubation, the cells were trypsinized and incubated with JC-1 at 37°C for 15 min. Subsequent to washing with JC-1 staining buffer twice, the cells were immediately analyzed by flow cytometry.

Hs578T cells (6×10⁵) were seeded in a 50-mm culture dish. On the following day, the cells were treated with AEXHP (0, 4, 8 or 12 mg/ml) for 48 h. Subsequent to trypsinization and washing with PBS, cells were fixed in 1 ml ice-cold 70% ethanol overnight at 4°C. The cells were centrifuged (1,000 × g, 5 min, 4°C), washed with cold PBS and treated with propidium iodide (PI)/RNase Staining Buffer (BD Pharmingen, San Diego, CA, USA) for 15 min at room temperature in the dark. The cell cycle distribution was then determined by flow cytometry. The percentage of cells in G₁, S and G₂/M phase was calculated using ModFit software v 3.2 (Becton Dickinson, San Diego, CA, USA).

Hs578T cells (2.25×10⁶) were seeded in a 100-mm culture dish. On the following day, the cells were treated with 0, 4, 8 or 12 mg/ml AEXHP. Following incubation for 24 or 48 h, the cells were harvested, washed with cold PBS and homogenized with radioimmunoprecipitation assay lysis buffer (TDYbio). Total protein was extracted and the protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific, Inc.). Subsequently, the proteins were mixed with 5X loading buffer (CWBIO, Beijing, China) and boiled for 5 min. Samples of 35 µg protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked for 2 h at room temperature with 5% fat-free milk (YiLi Inc., Huhehaote, China) or 5% bovine serum albumin (Amresco, Inc., Framingham, MA, USA) in 10 mM Tris-HCl, 0.1 M NaCl and 0.1% Tween-20, pH7.4 (TBST), and incubated with specific primary antibodies at 4°C overnight with gentle agitation. The membranes were washed with cold TBST and incubated with peroxidase-conjugated secondary antibody for 1 h. Visualization of the protein bands was accomplished using an Immobilon Western Chemiluminescent HRP Substrate (Millipore). The intensity of protein bands was measured using ImageJ software 1.48 (National Institutes of Health, Bethesda, MD, USA), normalized to that of β-actin and presented as a percentage of the control.

Values are expressed as the mean ± standard deviation of at least three independent experiments. Statistical analyses were performed using the two-tailed Student's t-test with SPSS 13.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

To evaluate the cytotoxic effects of AEXHP on Hs578T cells, the viability of cells (7.5×10³/well) treated with various concentrations of AEXHP (0–16 mg/ml) was determined using an MTT assay. The results showed that AEXHP decreased the number of viable cells in a dose- and time-dependent manner (Fig. 1A and B). The inhibition of Hs578T cell viability by AEXHP started relatively early at 12 h. At the highest concentration used (16 mg/ml), incubation with AEXHP for 12, 24 or 48 h significantly decreased the cell viability rate to 74.67±9.63, 52.07±10.89 and 22.21±8.30%, respectively, while there was no statistical difference between the control and the treatment group at the lowest concentration (4 mg/ml) at each time-point.

In order to investigate the difference in cytotoxicity/anti-proliferative effects of AEXHP on MCF-10A normal breast epithelial cells and Hs578T cells, each cell line was treated with 12 mg/ml AEXHP for 24 h. At a seeding density of 7,500 cells/well, the cell viability rate of Hs578T and MCF-10A decreased to 64.75±3.82 and 43.93±9.96%, respectively (Fig. 1C). There was a significant difference in the viability rate between these two cell lines (P<0.05). However, when the cell density was increased to 10,000 cells/well, the decreases in cell viability were similar between Hs578T and MCF-10A cells (71.67±10.60 and 72.41±9.80%, respectively, vs. control) (Fig. 1C).

In order to assess whether the anti-proliferative effects of AEXHP on Hs578T cells were due to induction of apoptosis, annexin V/PI double staining and flow cytometric analysis were used. After treatment with AEXHP (4, 8 or 12 mg/ml) for 24 h, the percentage of early apoptotic cells increased from 1.6% in the control group to 3.9, 5.4 and 8.6%, respectively, and the percentage of cells in late apoptosis increased from 8.0% in the control group to 12.2, 13.8 and 15.7%, respectively (Fig. 2A). At 8 and 12 mg/ml, AEXHP significantly increased the overall apoptotic rate. In conclusion, AEXHP induced apoptosis in Hs578T cells in a dose-dependent manner (Fig. 2B).

To assess whether the apoptosis of Hs578T cells was associated with the depletion of the Δψ_m, Hs578T treated with various concentrations of AEXHP were assessed by JC-1 staining and flow cytometric analysis. After treatment with AEXHP (4, 8 and 12 mg/ml) for 24 h, the percentage of cells with Δψ_m loss increased from 4.5% in the control group to 11.8, 14.4 and 19.6%, respectively, which was significant at the highest concentration (P<0.05) (Fig. 3A and B). Thus, AEXHP induced the depletion of the Δψ_m in Hs578T cells in a dose-dependent manner.

Apoptosis may proceed via two major pathways, namely the intrinsic and the extrinsic pathway (10), each of them leading to caspase-3 activation. The caspase family is the most prominent protease family in apoptosis (11), and is divided into two functional groups, namely the apoptosis initiators (caspase-8, −9 and −10) and the apoptosis executors (caspase-3, −6 and −7) (12).

Bax is a well-known pro-apoptotic protein, while Bcl-2 is an anti-apoptotic protein, and the Bcl-2/Bax ratio has a decisive role regarding the induction of apoptosis (13,14). To determine whether caspase-3, caspase-8, Bax and Bcl-2 proteins were involved in apoptosis of Hs578T cells induced by AEXHP, the levels of these proteins were assessed by western blot analysis. The results showed that after treatment with AEXHP (4, 8 and 12 mg/ml) for 24 h, levels of cleaved caspase-3 were increased to 1.70-, 1.81- and 1.84-fold of that of the control (P<0.05), while no significant impact on the levels of procaspase-3, cleaved caspase-8, procaspase-8, Bcl-2, Bax, and the Bcl-2/Bax ratio was observed (Fig. 4A and B). In conclusion, the results indicated that AEXHP induces apoptosis via the intrinsic pathway, but not the extrinsic pathway, as only an involvement of caspase-3 cleavage but no association with caspase-8, and Bcl-2/Bax ratio was found.

Cell cycle regulation is important for cell proliferation, and induction of cell cycle arrest is the major mechanism via which numerous anti-tumor drugs exert their anti-proliferative effects (15,16). To examine whether the anti-proliferative effects of AEXHP on Hs578T cells were associated with cell cycle arrest, their cell cycle distribution was determined. The results showed that AEXHP significantly affected the cell cycle distribution of Hs578T cells, leading to cell cycle arrest at S phase in a dose-dependent manner (Fig. 5B). While the S-phase population was 10.04% in the untreated group, treatment with 4, 8 and 12 mg/ml AEXHP increased it to 17.33, 41.91 (P<0.05 vs. control) and 52.52% (P<0.05 vs. control), respectively. Cell populations in G₁ or G₂/M phase shifted concomitantly to the changes detected in S phase (Fig. 5A), while there was no AEXHP dose-dependent decrease or increase in the G₁- or G₂/M-phase populations.

To explore the mechanisms by which AEXHP induced cell cycle arrest of Hs578T cells at S phase, western blot analysis was used to determine the expression of cell cycle regulatory proteins. The results showed that treatment with various concentrations of AEXHP (4, 8 or 12 mg/ml) for 48 h resulted in decreased expression of cyclin A and CDK2, as well as increased expression of cyclin E and p21^Cip1, as compared to the control group (Fig. 6A and B). The decreased expression of cyclin A and CDK2, and the increased expression of cyclin E and p21^Cip1, may have contributed to S-phase arrest of Hs578T cells induced by AEXHP.