MCF7, T47D, BT474, BT-549, MDA-MB-231 and MDA-MB-468 breast cancer cell lines, as well as a MCF10A normal breast epithelial cell line (control) were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were routinely cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, CA, USA) at 37°C in a 5% CO₂ incubator. MCF7, T47D and BT474 are estrogen receptor (ER) and progestogen receptor (PR)-positive cells, and human epidermal growth factor (HER)2-negative cells. BT-549, MDA-MB-231 and MDA-MB-468 are ER-, PR- and HER2-positive cells.

Isorhamnetin was purchased from Shanghai Tongtian Biotechnology Co., Ltd. (Shanghai, China). Perifosine, PD184352 and JSH-23 were purchased from Selleck Chemicals (Houston, TX, USA). EGF was purchased from Sigma-Aldrich (St. Louis, MO, USA). The antibodies for mouse monoclonal β-actin (cat. no. 3700; 1:1,000), rabbit polyclonal phosphorylated (p)-EGFR immunoglobulin (Ig)G (cat. no. 2234; 1:1,000), rabbit monoclonal EGFR (cat. no. 4405; 1:1,000), rabbit monoclonal PI3K (cat. no. 4249; 1:1,000), rabbit monoclonal p-Akt (S473; cat. no. 4060; 1:500), mouse monoclonal Akt (cat. no. 2920; 1:1,000), mouse monoclonal p-ERK1/2 (cat. no. 9106; 1:500), rabbit monoclonal ERK1/2 (cat. no. 4695; 1:1,000) and rabbit monoclonal cleaved caspase-3 (cat. no. 9664; 1:500) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA), and antibodies for rabbit monoclonal B cell lymphoma 2 (Bcl-2; cat. no. ab117115; 1:1,000), rabbit monoclonal Bcl-2-associated X protein (Bax; cat. no. ab32503; 1:1,000), rabbit monoclonal Bcl-extra large (xL; cat. no. ab32370; 1:2,000), rabbit monoclonal IκB (cat. no. ab32518; 1:1,000), rabbit monoclonal anti-NF-κB P65 antibody (cat. no. ab32536; 1:2,000) and rabbit polyclonal H3 (cat. no. ab1791; 1:2,000) were purchased from Abcam (Cambridge, MA, USA).

The cells were seeded into 96-well plates at a density of 5×10³ cells/well in 100 µl DMEM and placed in cell incubator for 12 h at 37°C in an atmosphere containing 5% CO₂. The cells were then treated with various concentrations of isorhamnetin (100, 33.3, 11.1, 3.7, 1.2, 0.4 and 0 µM) for 48 h, and cell proliferation rates were determined by adding 10 µl CCK-8 solution (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) prior to incubation at 37°C for 2 h. The absorbance was measured at a wavelength of 450 nm using a SpectraMax 190 Microplate Reader (Molecular Devices, LLC, Sunnyvale, CA, USA). For each assay, four parallel wells were included, and the half maximal inhibitory concentration (IC₅₀) was measured using the inhibition curve and presented as the mean of three independent experiments.

The cells were washed twice with ice-cold phosphate-buffered saline (PBS) and harvested using radioimmunoprecipitation (RIPA) buffer (Beyotime Institute of Biotechnology; 50 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, and 1% sodium deoxycholate, with protease and phosphatase inhibitors). The cell lysate was placed in RIPA for 30 min and centrifuged at a speed of 12,000 × g for 15 min, and the supernatant was then collected. Each aliquot of protein (10 µg) was separated by 12% SDS-PAGE and transferred to Hybond-C nitrocellulose membranes (GE Healthcare Biosciences, Pittsburg, PA, USA) in transfer buffer (192 mM glycine, 25 mM Tris, 2.5 mM SDS, and 10% methanol) (Sangon Biotech Co., Ltd., Shanghai, China). The membranes were blocked with 5% non-fat milk in Tris-buffered saline with 1% Tween-20 (TBST; Sangon Biotech Co., Ltd.) for 1 h at room temperature, incubated with the previously mentioned primary antibodies at 4°C overnight, then washed three times with TBST for 10 min prior to incubation with secondary horseradish peroxidase-conjugated anti-rabbit (cat. no. 7074; Cell Signaling Technology, Inc.) or anti-mouse IgG (cat. no. 7076; Cell Signaling Technology, Inc.) antibodies, and again washed three time with TBST for 10 min. The blots were developed with an Enhanced Chemiluminescence Plus Western Blotting Detection system (GE Healthcare Biosciences). The total protein was determined using the Bicinchoninic Acid method (Invitrogen Life Technologies; cat. no. 23235).

To determine the effects of isorhamnetin on cell apoptosis, the MCF7 or MDA-MB-468 cells were seeded at a density of 2×10⁵ cells/well in a 6-well plate, and incubated at 37°C overnight. The cells were treated with isorhamnetin or the inhibitors (perifosine, PD184352 and JSH-23) for 48 h, prior to being detached and washed with cooled PBS. The cells were collected by trypsin (Invitrogen Life Technologies) digestion and centrifugation at 300 × g for 3 min. The cells were then resuspended in binding buffer containing Annexin-V and propidium iodide (PI; Beyotime Institute of Biotechnology, Jiangsu, China) and incubated for 15 min in the dark at room temperature. Analysis was performed using a FACSCalibur analyzer (BD Biosciences, San Jose, CA, USA).

Protein expression was quantified using Image J software (version 1.31; Utrecht University, Utrecht, Netherlands) and expressed as the relative expression levels to the control group. P-values were calculated by comparison to the control group using analysis of variance with SPSS 19 (IBM SPSS, Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant result.

The inhibitory effects of isorhamnetin on breast cancer cells were determined using the CCK-8 method. As shown in Fig. 1A, isorhamnetin inhibited the proliferation of numerous breast cancer cells (IC₅₀, ~10 µM), including MCF7, T47D, BT474, BT-549, MDA-MB-231 and MDA-MB-468, whereas less inhibitory activity was observed in the MCF10A normal breast epithelial cell line (IC₅₀, 38 µM). These results indicated that isorhamnetin induces pronounced inhibitory effects on breast cancer cell lines (P<0.05), as compared to normal breast epithelial cell lines, which suggests that isorhamnetin may act on the activation pathway of cancer cells. The effect of isorhamnetin on cell apoptosis was subsequently determined using two breast cancer cell lines, MCF7 and MDA-MB-468. Isorhamnetin markedly promoted cell apoptosis of the MCF7 and MDA-MB-468 cell lines (Fig. 1B) as shown by the sum of early and late apoptotic cells, with increased apoptotic rates observed in the MCF7 cells, as compared with the MDA-MD-468 cells; these results were consistent with the IC₅₀ values of isorhamnetin that were determined for the two cell lines.

In order to investigate the inhibitory mechanism underlying the effects of isorhamnetin on breast cancer cells, the PI3K/Akt/mTOR and MEK/ERK signaling pathways, which are closely associated with cell proliferation and survival, were examined. As shown in Fig. 2A, the phosphorylation levels of Akt and mTOR were markedly decreased by isorhamnetin, as well as the phosphorylation levels of MEK1/2 and ERK1/2 both in MCF7 and MDA-MB-468 cells; however, those of their upstream signaling molecules, such as phosphorylated EGFR and PI3K (110α) remained unchanged. The expression of the components of the cell apoptosis signaling pathway, including Bax, Bcl-2, Bcl-xL and cleaved caspase-3 were also investigated. Bax expression was induced by isorhamnetin, whereas the Bcl-2 expression level was markedly downregulated, and a marginal decrease was observed in the expression level of Bcl-xL (Fig. 2B). Cleaved caspase-3 expression increased, implying that cell apoptosis was induced by isorhamnetin.

As isorhamnetin exhibits anti-oxidant effects, its impact on the downstream signaling molecule, NF-κB P65 was investigated, in order to identify whether these effects contributed to its anti-tumorigenic activity. As shown in Fig. 2C, the expression of cytosolic NF-κB P65 and its inhibitor, IκB were increased by isorhamnetin, whereas nuclear NF-κB P65 expression was decreased, suggesting that the nuclear translocation of NF-κB P65 is inhibited by isorhamnetin.

Akt and MEK inhibitors (perifosine and PD184352) were used to elucidate the mechanism of action of isorhamnetin (whether it is via the Akt or MEK signaling pathways) on breast cancer (Fig. 3). Perifosine is a potent pan-Akt inhibitor, which was used to block the Akt signaling pathway. Akt phosphorylation was inhibited by perifosine and partly inhibited by isorhamnetin (10 µM; Fig. 3A,). Following pretreatment of the MCF7 cells with perifosine prior to the addition of isorhamnetin, induction of cell apoptosis was only marginally increased by isorhamnetin, as determined by cleaved caspase-3 and Annexin-V/PI dual staining (Fig. 3C). These results demonstrate that blocking of Akt phosphorylation attenuated the effects of isorhamnetin.

Similarly, the MEK and ERK inhibitor, PD184352 was used to block the corresponding signaling pathways. As shown in Fig. 3B, PD184352 (1 µM) and isorhamnetin (10 µM) inhibited MEK1/2 phosphorylation and induced cell apoptosis. This was also determined by increased levels of cleaved caspase-3 expression and Annexin-V/PI dual staining (Fig. 3D). MCF7 cells were pre-treated with PD184352 prior to the addition of isorhamnetin, and the induction of cell apoptosis was only marginally increased following treatment with isorhamnetin. These results demonstrate that inhibition of the MEK signaling pathway also attenuates the effects of isorhamnetin.

EGF is a growth factor that binds to EGFR in order to induce the activation of downstream PI3K/Akt and MEK/ERK signaling pathways. Although EGFR phosphorylation did not change following treatment with isorhamnetin, the addition of EGF activated the Akt and MEK signaling pathways, and may be used to verify the effects of isorhamnetin. As shown in Fig. 4A, treatment with EGF markedly increased the phosphorylation levels of EGFR, Akt and MEK1/2, and these effects overrode the inhibitory effects of isorhamnetin on Akt and MEK1/2, thus inhibiting the cell apoptosis induced by isorhamnetin, as determined by decreased levels of cleaved caspase-3 expression.

The present study investigated whether the inhibition of NF-κB translocation contributed to cell apoptosis. An NF-κB inhibitor, JSH-23, was used to block the nuclear translocation of NF-κB, prior to treatment with isorhamnetin. As shown in Fig. 5, treatment with JSH-23 inhibited NF-κB translocation, but did not result in the apoptosis of MCF7 cells, either alone or in combination with isorhamnetin (as demonstrated by cleaved caspase-3 and Annexin-V/PI dual staining). These results suggested that inhibition of the NF-κB signaling pathway does not contribute to the induction of cell apoptosis by isorhamnetin.