The breast cancer cell lines MDA-MB-231 (ATCC no. HTB-26) and MCF-7 (ATCC no. HTB-22) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.) and supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.) under a humidified atmosphere containing 5% CO₂ and 95% air at 37°C. The culture medium was changed every other day.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) assay was applied to detect the cell viability (21). Briefly, samples of 100 µl breast carcinoma cells at a density of 50,000 cells/ml were seeded into 96-well plates. After 24- and 48-h treatment with astilbin (CAS no. 29838-67-3; obtained from Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China) at doses of 0–300 µM, MTT was added to each well (final concentration, 0.5 mg/ml). The cells were incubated for a further 4 h at 37°C in the dark, and then 100 µl dimethyl sulfoxide was added to solubilize the purple formazan crystals. A microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to detect the absorbance at a wavelength of 490 nm. The half maximal inhibitory concentration (IC₅₀) values were calculated by the SPSS version 16.0 software (SPSS, Inc., Chicago, IL, USA).

In order to examine the cell apoptosis, samples of 2 ml breast carcinoma cells at a density of 2×10⁵ cells/ml were seeded into 6-well plates. After 24-h exposure to astilbin at the doses of 50 and 200 µM, the cells were collected and stained with propidium iodide and Annexin V (Dead Cell Kit; EMD Millipore, Billerica, MA, USA) for 15 min at 25°C in the dark. A Muse™ Cell Analyzer from EMD Millipore was applied to analyze the fluorescence intensity.

For determination of the cell migration ability, a wound healing assay was performed. Briefly, the seeded cells were scraped with a p200 pipette tip, and then exposed to astilbin at the doses of 50 and 200 µM for 24 h. The distances traveled by the migrating cells were quantified using the ImageJ 1.48v software (National Institutes of Health, Bethesda, MD, USA; rsb.info.nih.gov/ij/download.html) to evaluate the cell migratory ability.

For intracellular ROS level determination, samples of 2 ml breast carcinoma cells at a density of 2×10⁵ cells/ml were seeded into 6-well plates, and exposed to astilbin at the doses of 50 and 200 µM for 12 h. Subsequently, cells were stained for 20 min with 2,7-dichlorofluorescein diacetate (Sigma-Aldrich; Merck KGaA) at 37°C in the dark. The changes in intracellular ROS levels were observed using fluorescent microscopy (magnification, ×20; CCD camera, Axio Observer Z1; Carl Zeiss AG, Oberkochen, Germany). The quantitative data were analyzed with the ImageJ software, and are expressed as the green fluorescence intensity.

For MMP determination, the treated cells were stained with 2 µM 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Sigma-Aldrich; Merck KGaA) for 15 min at 37°C in the dark. The changes in fluorescence from red to green were detected using fluorescent microscopy (magnification, ×20; CCD camera, Axio Observer Z1; Carl Zeiss AG). The quantitative data were analyzed with the ImageJ software, and are expressed as the ratio of red to green fluorescence intensity.

The experimental animal study was approved by the Ethics Committee of Changchun Medical College (approval no. CCMC2016-1201; Changchun, China). In total, 10 male BALB/c athymic nude mice (5-week-old; SCXK 2012-0001) were obtained from the Model Animal Research Center of Nanjing University (Nanjing, China). The mice were maintained on a 12-h light/dark cycle at 23±1°C with water and food available ad libitum. A sample of 0.15 ml MCF-7 cell suspension at a density of 2×10⁸ cells/ml was subcutaneously injected into the right-side waist of each nude mouse. After 4 days, the mice were randomly divided into two groups (n=5 each), and intraperitoneally injected with astilbin (20 mg/kg; 0.3 ml/mouse) or normal saline (0.3 ml/mouse), respectively, every other day for 14 days. The tumor dimensions were monitored throughout the experiment. The tumor volume (mm³) was estimated using the following equation: Volume = length × (width)² × 0.5. Finally, the mice were sacrificed by administration of 200 mg/kg pentobarbital. Liver, spleen, kidney and tumor tissues were carefully dissected from each mouse for western blot analysis. The protocol of the animal experiments was followed as described in previous studies (22–24).

Blood was collected from the caudal vein of each mouse prior to euthanasia. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum were measured using commercial diagnostic kits (Nanjing Jiancheng Institute of Biotechnology Co., Ltd., Nanjing, China).

The liver, spleen and kidney tissues were fixed in 4% paraformaldehyde and subjected to histopathological examination using hematoxylin and eosin staining, as described in our previous study (15).

Samples of 4×10⁵ cells per well were plated into 6-well plates, and treated with astilbin at the doses of 50 and 200 µM for 24 h. The treated cells and collected tumor tissues from the nude mice were lysed by radioimmunoprecipitation assay buffer containing 1% protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA). Subsequent to protein concentration detection, 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to separate the protein samples (40 µg), which were further transferred electrophoretically onto 0.45-µm nitrocellulose membranes (Bio Basic, Inc., Markham, ON, USA). The membranes were blotted with primary antibodies, including B-cell lymphoma 2 (Bcl-2; cat. no. 3498), Bcl-2-associated X protein (Bax; cat. no. 14796), cleaved caspase-3 (cat. no. 9661), −8 (cat. no. 8592) and −9 (cat. no. 9509) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; cat. no. 5174) (all purchased from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C for 12 h. The dilution of all primary antibodies was 1:2,000. After three washes with Tris-buffered saline/Tween-20 buffer, the membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (cat. nos. 7074 and 7076; Santa Cruz Biotechnology, Inc.) at a dilution of 1:3,000 for 4 h at room temperature. Chemiluminescence was performed using ECL detection kits (GE Healthcare Life Sciences, Little Chalfont, UK). ImageJ software was used to detect the intensity of the bands via scanning densitometry.

All experimental data in the present study are expressed as the mean ± standard deviation. The statistical data were analyzed using one-way analysis of variance, followed by post hoc multiple comparisons (Duncan's multiple range test) using the SPSS version 16.0 software (SPSS, Inc., Chicago, IL, USA). A value of P<0.05 was considered to denote a statistically significant difference.

Astilbin reduced the viability of both MDA-MB-231 and MCF-7 cells in a dose-dependent manner (P<0.001 at 300 µM; Fig. 2A). The half maximal inhibitory concentration (IC₅₀) values of astilbin for MDA-MB-231 and MCF-7 cells for a 24-h exposure were approximately 167.9 and 191.6 µM, respectively (Fig. 2A). The percentages of cell apoptosis after 24-h exposure to 50 µM astilbin were >19.1% (P<0.01) and >24.5% (P<0.001) in MCF-7 cells and MDA-MB-231, respectively (Fig. 2B). Furthermore, the effects of astilbin on the migration ability of the breast carcinoma cells were investigated using a wound healing assay. In contrast to the non-treated cells, the cells treated with 24-h exposure to astilbin at the doses of 50 and 200 µM exhibited significantly reduced migration into the wound area (Fig. 2C), indicating the suppressed migration abilities of the breast carcinoma cells.

Intracellular levels of ROS not only influence mitochondrial function, but also serve an important role during cell apoptosis (14). Compared with the non-treated cells, the treated cells exhibited an enhanced green fluorescence intensity (Fig. 3A), suggesting the potential role of astilbin in promoting ROS production. In addition, treatment with astilbin at 200 µM caused a statistically significant accumulation of ROS by >5-fold in MDA-MB-231 and MCF-7 cells (P<0.001; Fig. 3A).

JC-1 staining was also performed to investigate the regulatory effect of astilbin on the MMP in the breast carcinoma cells, which is an indicator of mitochondrial function. Incubation with astilbin for 12 h strongly reduced the MMP in breast carcinoma cells, as indicated by the increased green fluorescence intensity and reduced red fluorescence intensity (Fig. 3B). Compared with the control cells, astilbin resulted in an approximately 50% reduction in the ratio of red/green fluorescence intensity in the treated breast carcinoma cells (P<0.001; Fig. 3B).

Members of the caspase family, which are considered as critical participants in intrinsic and extrinsic mitochondrial signaling, were analyzed in the present study via western blot assay. It was observed that astilbin significantly increased the expression levels of cleaved caspases-3, −8 and −9 in MDA-MB-231 and MCF-7 cells after 24-h exposure (P<0.05; Fig. 4). Furthermore, Bax and Bcl-2 levels were examined, since the ratio of Bax and Bcl-2 serves as an important index for mitochondrial function (15). Compared with the control cells, 24-h exposure to astilbin resulted in a significant increase in Bax expression levels and marked suppression of Bcl-2 expression levels (P<0.05; Fig. 4). All these data conclusively confirmed that astilbin induced intracellular toxicity in breast carcinoma cells through the modulation of mitochondrial function.

In tumor xenograft male BALB/c nude mice, 20 mg/kg astilbin was intraperitoneally injected every other day for 14 days. The tumor growth was evidently suppressed by astilbin as compared with that observed in the control mice (Fig. 5A and B). The inhibitory activities of astilbin on tumor growth became apparent from day 7, and caused >6-fold inhibition of the MCF-7 ×enograft tumor growth by the day 15 (P<0.05; Fig. 5C). However, astilbin had no significant influence on the body weight of mice (Fig. 5D), serum levels of AST and ALT (Fig. 5E), or organ function, including the liver, spleen and kidneys of the mice, (Fig. 5F), suggesting its safety for mouse treatment.

The expression levels of anti- and pro-apoptotic proteins in the tumor tissues were also measured. The results demonstrated that 14-day astilbin treatment significantly enhanced the expression levels of Bax and cleaved caspase-3, −8 and −9, and reduced the expression levels of Bcl-2 in the tumor tissues of nude mice (P<0.01; Fig. 6).