The breast cancer cell lines MCF-7, MDA-MB-231 and MCF-10 were purchased from Cell Applications, Inc. (San Diego, CA, USA). The cells were cultured in cell growth medium supplemented with 100 IU/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified incubator with 5% carbon dioxide (CO₂) and 95% air.

The cell growth medium was replaced with serum-free medium for an additional 24-h culture prior to further treatments. Cells were suspended at a density of 2×10⁶ cells/ml for experiments. In the mechanism experiments, cells treated with serum-free medium alone served as the control group. The cells were treated with 20 or 30 μM baicalin in Dulbecco’s modified Eagle’s medium for 48 h. The concentrations of baicalin were selected as previously described (10-13,18).

The present study was approved by the Ethics Committee for Animal Experimentation of the School of Life Science and Technology, Harbin Institute of Technology. All animal experiments were performed according to the Guidelines for the care and use of experimental animals approved by the Heilongjiang Province People’s Congress (http://www.nicpbp.org.cn/sydw/ CL0249/2730.html). Female BALB/c nude mice, 5 weeks old and weighing ~15 g, were purchased from the Harbin Veterinary Research Institute (Harbin, China). The animals were housed in a clean and ventilated environment under standard laboratory conditions (temperature 22±1°C, relative humidity 50-70%, and a 12-h light/dark cycle). Standard food and water were provided ad libitum throughout the experiments. The animals were allowed to acclimatize to their surroundings over 7 days to eliminate the effect of stress prior to the initiation of the experiments, and then randomly divided into three groups (control, 100 and 200 mg/kg groups; n=6 per group) for the following tumorigenicity and baicalin treatment experiments.

MDA-MB-231 cells (1×10⁵/injection) were suspended in 100 μl phosphate-buffered saline (PBS) containing 50% Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) and injected into the mammary fat pad of 4-5-week-old female nude mice. One week later, the control mice received PBS (1 ml/kg, once daily i.p.), while 100 and 200 mg/kg group mice received daily i.p. injections of baicalin (>95% purity; Sigma-Aldrich; Merck KGaA, St. Louis, MO, USA) at doses of 100 and 200 mg/kg, respectively. The concentrations of baicalin were selected as previously described (10-13,18). Tumor size was measured every 5 days for 4 weeks, starting 5 days after tumor cell implantation. The tumor volume was calculated according to the following formula: V = L x W²/2, where V, volume (mm³); L, length (mm); and W, width (mm).

Cells were cultured using the abovementioned method, diluted to 2×10⁶ cells/ml, seeded into 96-well plates (100 μl/well) and incubated for 24 h, followed by the addition of 20 or 30 μM baicalin and further incubation for 72 h. According to the manufacturer’s recommendations, 20 μl 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazoliumbro-mide solution (5 mg/ml in PBS; Sigma-Aldrich; Merck KGaA) was added to each well and incubated with cells under standard conditions for 4 h. Subsequently, the formazan crystals in each well were dissolved in dimethyl sulfoxide, after removal of the medium. Finally, the optical density at 490 nm was measured using an enzyme-linked immunosorbent microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The results are expressed as the mean of triplicate experiments.

Untreated cells and those treated with baicalin were dissociated and washed with PBS for cell cycle analysis. The cells were incubated in PBS containing 500 U/ml RNase (Sigma-Aldrich; Merck KGaA) at 37°C for 15 min, incubated with 50 μg/ml propidium iodide (PI) at 4°C in the dark for 2 h, and analyzed to determine the percentage of 10,000 cells in each of three cell cycle compartments (G0/G1, S and G2/M) using a fluorescence-activated cell analyzer (Becton-Dickinson, Mountain View, CA, USA). Modifit LT 2.0 software was used to perform data analysis, and CELLQuest software (both from Becton-Dickinson) was used to quantify the percentage of cells in each compartment.

To measure cell invasion, 8-μm pore 24-well Matrigel invasion chambers (Corning Inc., Corning, NY, USA) were used according to the manufacturer’s instructions. A total of 2×10⁴ cells were seeded into each well. DMEM with 0.1% FBS was added to the upper chamber, while DMEM supplemented with 10% FBS was added to the lower chamber well to promote cell invasion. After 24 h of incubation at 37°C with 5% CO₂, non-invading cells were removed from the top well, while migrated cells were quantified by capturing photographs of 6 independent visual fields under an Olympus BX51 microscope (Olympus Co., Tokyo, Japan), and was stained with 0.1% crystal violet solution. Independent experiments were repeated at least three times.

A scratch wound healing assay was performed to evaluate the migration of untreated cells and cells treated with baicalin. In brief, MCF-7 and MDA-MB-231 cells were seeded in 6-well plates and cultured with DMEM supplemented with 10% FBS. After reaching confluence, each well was scratched with a 200-μl pipette tip. After 12 and 24 h of incubation, 6 wound healing areas were imaged and the distance between the two edges was analyzed by ImageJ software, version 1.48 (National Institutes of Health, Bethesda, MD, USA).

The pRL-TK and recombinant NF-ĸB luciferase reporter plasmids (Thermo Fisher Scientific, Inc., Waltham, MA, USA) were co-transfected into MCF-7 and MDA-MB-231 cells for measurement of NF-ĸB reporter activity. Transfected cells were harvested 48 h after transfection, and luciferase activity was determined by Fluoroskan Ascent FL (Thermo Fisher Scientific, Inc.) configured for dual assays. The relative luciferase activity was normalized to Renilla luciferase activity. All experiments were performed in triplicate wells.

At day 30 of baicalin treatment, the mice were anesthetized with intraperitoneal injection of 10% chloral hydrate (400 mg/kg body weight). Blood samples were collected from the abdominal aorta using syringes, and centrifuged for 30 min (3,000 × g at 4°C). Serum samples were stored at -80°C until use. The mice were then euthanized by cervical dislocation. To determine cytokine levels in vitro, MCF-7 and MDA-MB-231 cells were treated with or without baicalin using the method described above. The supernatants of culture plates were harvested from each well. TNF-α and IL-1β levels in the serum and culture supernatants were measured using mouse TNF-α and IL-1β ELISA kits (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

MCF-7 and MDA-MB-231 cells and 0.3-g tumor samples from mice were treated with RIPA lysis buffer supplemented with protease inhibitor cocktail and protein phosphatase inhibitor. The protein concentration was quantified using a DC protein kit (Bio-Rad Laboratories). Samples were then separated, transferred to PVDF membranes, and incubated with 1:1,000 dilution primary antibodies overnight at 4°C. The primary antibodies used were anti-phospho-NF-ĸB-p65 (Ser536) (rabbit mAb, catalog no. 3033), anti-phospho-IKKα/β (Ser176/180) (rabbit mAb, catalog no. 2697) and anti-IĸBα (rabbit mAb, catalog no. 4812). The anti-total NF-ĸB-p65 (rabbit mAb, catalog no. 8242), anti-total-IKKβ (rabbit mAb, catalog no. 2370) and anti-β-actin antibodies (rabbit mAb, catalog no. 4970) were used as loading controls. On the following day, the membranes were incubated with secondary antibody (anti-rabbit IgG, catalog no. 4414) for 2 h at room temperature. All antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The bands were visualized using an ECL Plus western blotting detection system (GE Healthcare, Milwaukee, WI, USA).

Total RNA was extracted from MCF-7 and MDA-MB-231 cells and xenograft tumor tissues using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Grand Island, NY, USA) according to the manufacturer’s instructions. Equal amounts (1 μg) of total RNA were used to perform reverse transcription using PrimeScript™ RT reagent kit (Takara, Tokyo, Japan). For the determination of the expression of various genes in MCF-7 and MDA-MB-231 cells and xenograft tumor tissues, RT-qPCR was performed using the following primers: CCND1, forward, 5′-AGAAGCTGTGCATCTACACCGACA-3′ and reverse, 5′-TGATCTGTTTGTTCTCCTCCGCCT-3′; BCL2, forward, 5′-TGAGCAGAGTCTTCAGAGACAGCC-3′ and reverse, 5′-ATGTGTGTGGAGAGCGTCAACC-3′; BIRC2, forward, 5′-CCCAAAGACTTTTCCCAGGTCCC-3′ and reverse, 5′-ACTGAGCTTCCCACCACAGGCA-3′; BIRC3, forward, 5′-GAATACTCCCTGTGATTAATGCTGCCGTGG-3′ and reverse, 5′-TCTCTTGCTTGTAAAGACGTCTGTGTCTTC-3′. The ACTB (β-actin) control primers were 5′-CGTTGCTATCCAGGCTGTGCTA-3′ and 5′-CCAGGTCCAGACGCAGGATGGC-3′. Quantification of gene expression was determined by comparative quantity, using ACTB gene expression as internal control. The PCR conditions were denaturation at 94°C for 45 sec, annealing at 57°C for 30 sec, and extension at 72°C for 1 min, for 40 cycles.

Data are expressed as mean ± standard error of the mean and were analyzed using GraphPad Prism version 5.0 (GraphPad Software, La Jolla, CA, USA). All calculated significant differences were based on one-way analysis of variance and the post hoc Tukey’s test. For direct comparisons between two groups, the Student’s t-test was used. P<0.05 was considered to indicate statistically significant differences.

To evaluate the effect of baicalin on breast cancer, cell proliferation was assessed. The cells were manually counted in 6 wells per group. The results demonstrated that baicalin reduced cell proliferation from 48 to 72 h in MCF-7 and MDA-MB-231 cells, indicating that this compound can inhibit breast cancer cell proliferation (Fig. 1A). In addition, the effect of baicalin on non-tumorigenic normal breast epithelial cells was assessed by comparing the proliferation of MCF-7 and MCF-10 cells. Baicalin did not affect the proliferation of MCF-10 cells, indicating that this compound mediated growth arrest of breast cancer cell lines without affecting the non-tumorigenic normal breast epithelial cells (Fig. 1A). To further elucidate the mechanism of action of baicalin in breast cancer development, after 48 h of treatment, cell cycle analysis was performed by flow cytometry. In MCF-7 cells, the cell phase distributions before and after treatment with 20 and 30 μM baicalin were as follows: G0/1 phase, 49.53±0.62 vs. 55.34±1.74 and 63.52±3.64%; S phase, 31.75±1.24 vs. 25.73±2.79 and 25.51±3.61%; and G2/M phase, 18.71±0.45 vs. 17.93±1.87 and 17.66±2.2, respectively. In MDA-MB-231 cells, the cell phase distributions before and after treatment with 20 and 30 μM baicalin were as follows: G0/1 phase, 42.96±2.01 vs. 53.1±1.74 and 64.96±3.33%; S phase, 29.53±1.12 vs. 24.17±2.71 and 24±2.29%; and G2/M phase, 17.51±1.79 vs. 18.17±1.74 and 13.41±2.48%, respectively (Fig. 1B and C). These results indicate that the proportion of cells in the S phase was significantly reduced following treatment with baicalin, whereas the proportion of cells in the G0/1 phase was significantly increased, suggesting that baicalin induces G1/S arrest. Compared with MCF-7 cells, baicalin did not affect the cell cycle distribution of MCF-10 cells (Fig. 1B and C).

The Transwell assay was employed to evaluate the effect of baicalin on the invasion of MCF-7 and MDA-MB-231 cells. The cells were manually counted in 6 wells per group. The mean number of invasive cells, as determined by microscopy, was 100±19.5, 43±13.7 and 41±13.3 in untreated MCF-7 cells, and those treated with 20 and 30 μM baicalin, respectively. The mean number of invading cells, as determined by microscopy, was 100±13.6, 54±15.3 and 44±16.3 in untreated MBA-MB-231 cells, and those treated with 20 and 30 μM baicalin, respectively. Therefore, the number of invading cells in the treatment groups was significantly reduced compared with that in the untreated control group (Fig. 2A and B).

Next, a wound healing assay was performed to explore the effects of baicalin on the migration of MCF-7 and MDA-MB-231 cells. The cells were manually counted in 6 wells per group. After 24 h, the number of migrating MCF-7 cells was 115±16.3, 67±7.7 and 64±10.1 in untreated cells, and those treated with and 20 and 30 μM baicalin, respectively. The number of migrating MDA-MB cells was 103±12.6, 52±10.6 and 44±7.3 in untreated cells, and those treated with 20 and 30 μM baicalin, respectively. Therefore, the number of migrating cells in the treatment groups was significantly reduced compared with that in the untreated control group (Fig. 2C and D).

To further explore the effects of baicalin on breast cancer growth, an MBA-MB-231 xenograft mouse model was employed, using 6 mice per group. After 25 days, the tumor volumes were smaller in the MBA-MB-231 cell xenograft mice treated with 100 and 200 mg/kg baicalin compared with the untreated group (124.77±38.33 and 116.22±35.64 vs. 363.63±47.63 mm³, respectively; Fig. 3A-C). In the control group, the longest diameter of the largest subcutaneous tumor was 15.62 mm. The tumor weights were also reduced in the 100 and 200 mg/kg baicalin treatment groups compared with the untreated group (0.12±0.03 and 0.10±0.05 vs. 0.25±0.02 g, respectively; Fig. 3B and D). No multiple subcutaneous tumors or peritonitis were observed in mice after being injected with MDA-MB-231 cells or baicalin.

To investigate whether baicalin modulates the inflammatory process by regulating the secretion of cytokines, the serum levels of TNF-α and IL-1β were measured in vitro and in vivo by ELISA. The cells were manually counted in 6 wells per group; the number of mice per group was n=6. As shown in Fig. 4A and B, high levels of the pro-inflammatory cytokines TNF-α and IL-1β were detected in the medium of MCF-7 and MBA-MB-231 cells in the control group. By contrast, the levels of these cytokines were significantly lower in the baicalin-treated groups (20 and 30 μM) compared with the untreated group. However, there was no significant difference in the cytokine levels between mice treated with 20 and those treated with 30 μM baicalin. Similar results were observed in serum samples obtained from mice on day30 following treatment with 100 or 200 mg/kg baicalin (Fig. 4C).

Based on the key role of NF-κB in the regulation of tumor-associated inflammation and cancer progression, we next investigated the association between baicalin and NF-κB. The cells were manually counted in 6 wells per group, and the number of mice per group was n=6. In a dual luciferase reporter assay, a 3-fold lower luminescence intensity was observed in cells treated with 20 and 30 μM baicalin compared with the control group in vitro, indicating that the activation of NF-κB was inhibited by baicalin (Fig. 5A). Western blot analysis further confirmed that NF-κB was inhibited by baicalin in breast cancer cells in vivo and in vitro. The phosphorylation levels of IκB kinase β (IKKβ) and NF-κB-p65 were reduced, whereas the expression of IκBα, an essential NF-κB inhibitor, was markedly upregulated in response to baicalin treatment (Fig. 5B and C).

NF-κB also regulates oncological behavior. The expression of cyclin D1 (CCND1), BCL2, BIRC2 (cIAP1) and BIRC3 (cIAP2) were examined by RT-qPCR. The results demonstrated that baicalin reduced the expression of these genes, indicating that baicalin may suppress cell proliferation by inhibiting NF-ĸB-induced expression of CCND1, and suppress cell invasion and migration by inhibiting NF-ĸB-induced expression of BCL2, BIRC2 and BIRC3 (Fig. 5D).