3-(4,5)-dimethlythiahiazo-z-y10-3,5-diphenytetrazoliumromide (MTT) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Annexin V-FITC/PI was manufactured by Beijing Zhaungmeng Science and Technology Limited Company (Beijing, China). Hoechst 33258 dye was obtained from Sigma-Aldrich. Total flavonoids were dissolved in DMSO and diluted with culture media immediately prior to use. Primary and secondary antibodies were manufactured by Beijing Boaosen Biotechnology Limited Company (Beijing, China). The T. chinensis flavonoids were prepared as previously described (15) with a purity of ~68%.

MCF-7 breast cancer cells maintained at the Medical Genetics Department of Beijing Cancer Institute were cultured in RPMI-1640 (Gibco, ThermoFisher Scientifc, Inc., Waltham, MA, USA) medium supplemented with 10% fetal bovine serum (FBS), 100 µM/ml gentamicin at 37°C in an atmosphere with 5% CO₂. Cells were subcultured and passaged at ~70–80% confluency. Cells in the logarithmic phase were used in all experiments. Total flavonoids were extracted from T. chinensis. Stock solutions, kept at −20°C, were diluted to the final concentrations with culture medium prior to use. The final DMSO concentration was below 0.2% in all experiments.

The effect of total flavonoids on cell growth was evaluated by using an MTT assay. Briefly, cells were seeded at 10⁵ cells/ml in 96-well plates for the assay. After culturing with flavonoids at different concentrations (0, 0.0991, 0.1982, 0.3964, 0.7928 or 1.5856 mg/ml) for various times (24, 48 or 72 h), 20 µl of MTT was added to each well. Plates were incubated for another 4 h at 37°C, and then the medium was aspirated by pipetting and replaced with 100 µl/well DMSO in order to dissolve the formazan produced by viable cells exposed to MTT. The absorbance reflecting the cell growth was measured at 490 nm with a microplate reader. All data were obtained from 4 independent experiments and expressed as the mean ± standard deviation.

Morphological changes were detected with Hoechst dye staining. Cells were seeded (10⁵ cells/ml) on coverslips overnight and then treated with flavonoids at different concentrations (0, 0.0991, 0.1982, 0.3964, 0.7928 or 1.5856 mg/ml) for 24 h. Thereafter, the coverslips were washed with PBS and fixed with 4% paraformaldehyde for 1 h. After washing the cells with PBS, images were captured with DIC software (Nikon, Tokyo, Japan). The cells on the coverslips were incubated with Hoechst 33258 staining solution for 30 min, washed 3 times for 2 min with PBS and mounted on microscope slides. A fluorescence microscope (Nikon) was used to capture images using an excitation wavelength of 340 nm. The entire experiment was performed at room temperature.

Scanning electron microscopy was used for observation of morphological changes in apoptotic cells. MCF-7 cells were seeded in a 24-well plate at the density of 10⁵ cells/ml. Overnight, cells were treated with culture medium or total flavonoids at 0, 0.0991, 0.1982, 0.3964, 0.7928 and 1.5856 mg/ml. A total of 24 h later, the supernatant was discarded, and the cells were washed with PBS, fixed with glutaraldehyde for 24 h, washed 3 times with PBS for 1 min and then dehydrated in a gradient of alcohol solutions for 1 min at each concentration. Finally, the cells were soaked in tert-butyl alcohol twice for 10 min each time, dried with a vacuum pump for 24 h and metal spray-coated for 90 sec. The prepared specimen was observed under a scanning electron microscope.

MCF-7 cells were seeded in a 6-well plate at 3×10⁵ cells/well in RPMI-1640 with 10% FBS. After 24 h, cells were treated with flavonoids at a concentration of 0, 0.0991, 0.1982, 0.3964, 0.7928 or 1.5856 mg/ml. Untreated cells served as the control group. After 24 h, supernatants were discarded, and the cells were fixed with 1% paraformaldehyde for 24 h. After washing with PBS twice, cells were stained with hematoxylin for 15 min, differentiated in hydrochloric acid alcohol for 10 sec, treated with ammonium hydroxide for 20 sec and stained with eosin for 10 min, with rinses under running water after each step. Subsequently, the cells were subjected to gradient alcohol dehydration, and dimethylbenzene was used for rendering the slides transparent. Finally, the cells were mounted with resin and observed under a light microscope.

Laser scanning confocal microscopy was used for observation of cell morphological changes during apoptosis. MCF-7 cells were seeded in 6-well plates in RPMI-1640 media containing 10% FBS. After 24 h, the cells were exposed to flavonoids at concentrations of 0, 0.0991, 0.1982, 0.3964, 0.7928 and 1.5856 mg/ml for 24 h. The supernatant was discarded, and the cells were washed 3 times with PBS for 2 min each time. After incubation with 5 µl Annexin V and 10 µl PI for 10 min in the dark, cells were washed with PBS for 2 min and images were immediately captured under a laser scanning confocal microscope.

MCF-7 cells grown in RPMI medium with 10% FBS were treated with flavonoids at different concentrations. After 24 h, cells were harvested and disrupted in RIPA lysis buffer to extract total cellular proteins. The protein content was determined with a BAC protein determination kit (Wuhan Boshide Bio-engineering Limited Company, Wuhan, China). Total cellular proteins (50 µg) were separated in 15% SDS-PAGE gels and transferred into PVDF membranes (Sigma-Aldrich) with a wet transfer system (Bio-Rad Laboratories). Membranes were blocked in 5% non-fat milk for 1 h at room temperature with 5% fat-free milk dissolved in PBST buffer. Thereafter, the membranes were probed with β-actin (cat. no. ZA109; Beijing Zoman Biotechnology Co., Ltd., Beijing, China), bcl-2 (cat. no. bs-0032R; BIOSS, Beijing, China), FasL (cat. no. bs-0216R; BIOSS), caspase-3 (cat. no. ZA135; Beijing Zoman Biotechnology Co., Ltd.), caspase-9 (cat. no. ZA137; Beijing Zoman Biotechnology Co., Ltd.), p53 (cat. no. ZA120; Beijing Zoman Biotechnology Co., Ltd.) and NF-κB (cat. no. ZA131; Beijing Zoman Biotechnology Co., Ltd.) primary antibodies in TBST buffer containing 0.1% Tween-20 at 1:1,000 dilution for 24 h at 4°C, followed by exposure to horseradish peroxidase-conjugated secondary antibodies (Beijing Boaosen Corporation, Beijing, China) for 90 min at temperature. The protein levels were visualized using a Western blot detection system, and immunoreactivities were detected with an enhanced colorimetric detection kit (Amplified Alkaline Phosphatase Goat Anti-Rabbit Immun-Blot Assay Kit, Bio-Rad Laboratories, Inc., Hercules, CA, USA).

An MTT assay was performed to examine the effect of total flavonoids on cell growth. MCF-7 cells were treated with different concentrations (0, 0.0991, 0.1982, 0.3964, 0.7928 and 1.5856 mg/ml) of total flavonoids for 24, 48 and 72 h. As presented in Fig. 1, the survival of MCF-7 cells reduced in a time-dependent and dose-dependent manner. At 72 h, the maximum inhibition ratio reached up to 81.31% at the drug concentration of 1.5856 mg/ml (Table I).

In the control group, normal fusiform-shaped cells were connected and radiated outward, adhering tightly to the cover glass. With a low concentration of flavonoids (0.0991 mg/ml), the shape of cells started to change. Upon exposure to increased flavonoid concentrations (0.1982 and 0.3964 mg/ml), the cells rounded up with loosened cell junctions and appeared to be vacuolated. At high concentrations of flavonoids (0.7928 and 1.5856 mg/ml), fractured junctions, swelled nuclei, plasmolysis and a surge in the number of desquamated cells was observed (Fig. 1).

To examine the cell death induced by total flavonoids, the nuclear morphology of dying MCF-7 cells stained with Hoechst 33258 dye was observed. In the control group, the nuclei of cells had a regular shape and showed a uniform distribution of low-density fluorescence. In the group exposed to a low concentration of flavonoids (0.0991 mg/ml), the nuclei of a proportion cells appeared darker than those of normal cells, indicating apoptotic cell death. In groups treated with flavonoids in the middle of the concentration range (0.1982 and 0.3964 mg/ml), cell numbers gradually decreased, while the proportion of cells with hyperchromatic nuclei increased. With high concentrations of flavonoids (0.7928 and 1.5856 mg/ml), MCF-7 cell numbers fell sharply, fragmented nuclei appeared and typical apoptotic characteristics became more apparent (Fig. 2).

MCF-7 cells in the control group were firmly adherent and covered with abundant microvilli. Those cells were connected tightly with neighboring cells and extended in all directions. In the low flavonoid concentration group (0.0991 mg/ml), cells with gap junctions shrank and showed decreased surface microvilli. In the middle concentration group (0.1982 and 0.3964 mg/ml), characteristics of apoptosis became marked. With high concentrations of flavonoids (0.7928 and 1.5856 mg/ml), the microvilli nearly completely disappeared, and cell membranes collapsed (Fig. 3).

In the control group, cells were in good condition and able to grow adhering to the cover glass. The nucleoplasm was uniformly stained, and mitoses could be observed. The nucleolus and nuclear membrane profiles also could be seen. At a low concentration (0.0991 mg/ml), treatment with total flavonoids halted cell division. With flavonoids in the middle range of concentrations (0.1982 and 0.3964 mg/ml), intercellular spaces dilated. However, cells with condensed cytoplasm maintained their plasma membrane integrity. The cells shrank and appeared increasingly deformed or dehydrated. At high concentrations of flavonoids (0.7928 and 1.5856 mg/ml), adherent cells were drastically reduced in number and cells were no longer intact. Cells treated with high flavonoid concentrations (0.7928 and 1.5856 mg/ml) were more hyperchromatic than the cells of the middle concentration group (0.1982 and 0.3964 mg/ml). The HE result revealed shows swollen nucleoli and fractured junctions, which was in accordance with what DIC and scanning electron microscopy results demonstrated (Fig. 4).

In the control group, MCF-7 cells were not readily stained by Annexin V or PI. In the low flavonoid concentration group (0.0991 mg/ml), cells in the early stage of apoptosis were seen. Plasma cell membranes labeled green by Annexin V appeared gradually. In the middle concentration range of flavonoids (0.1982 and 0.3964 mg/ml), the integrity of the cytomembrane was no longer intact and stained green, while the nucleus was dyed red. In the high flavonoid concentration group (0.7928 and 1.5856 mg/ml), both the cytomembrane and nucleus were fragmented (Fig. 5).

In order to study the mechanism of flavonoid-induced apoptosis in MCF-7 cells, the expression of caspases, fasL, p53 and bcl-2 were determined. After 24 h of exposure to flavonoids, the expression of cleaved caspase-3 and caspase-9 increased in a dose-dependent manner. Bcl-2 is a crucial determinant of cell apoptosis, and a decrease in its expression was observed in the flavonoid-treated cells. This observation suggests that an intrinsic apoptotic signaling contributed to the damaging effects of flavonoids on MCF-7 cells. Total flavonoids also down-regulated the expression of NF-κB, which in turn could down-regulate the expression of bcl-2. By contrast, the expression levels of p53 and fasL were not markedly changed. These findings suggest that induction of the mitochondrial pathway is crucial for flavonoid-induced apoptosis of MCF-7 cells (Fig. 6).