Baicalin (purity >99%) was obtained from Zelang Medical Technology Co. (Nanjing, China). Crystal violet was purchased from Sigma (St. Louis, MO, USA). Vectastain ABC kit and liquid DAB+ Substrate Chromogen system for immunohistochemistry were purchased from Vector Laboratories Inc. (Burlingame, CA, USA) and Dako (Carpinteria, CA, USA), respectively. RPMI-1640 medium, Dulbecco's modified Eagle's medium (DMEM), trypsin-EDTA, phosphate-buffered saline (PBS) and penicillin/streptomycin were the products of Hyclone Laboratories Inc. (Los Angeles, CA, USA). Fetal bovine serum (FBS) was purchased from Gibco (Grand Island, NY, USA). Antibodies used in this study were anti-E-cadherin, anti-claudin, anti-vimentin, anti-N-cadherin, anti-β-catentin, anti-Snail and anti-Slug from Cell Signaling Technology (New England Biolabs, Ipswich, MA, USA); anti-Ki67 (rabbit monoclonal), secondary antibody (rabbit monoclonal IgG) from Abcam (Cambridge, UK); anti-GAPDH from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-F-actin-Green 488, and Alexa Fluor 594 (goat-anti-rabbit IgG) from Molecular Probes (Oregon, Eugene, OR, USA).

Human breast cancer cell line MDA-MB-231 and mouse mammary cancer cell line 4T1 were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in DMEM and RPMI-1640 medium containing 10% FBS and 1% penicillin/streptomycin, respectively. Both of the cell lines were cultured in a humidified atmosphere with 5% CO₂ at 37°C.

Cell viability was determined by crystal violet assays. MDA-MB-231 and 4T1 breast cancer cells were harvested with 0.05% trypsin-EDTA and seeded in 24-well plates (Corning, MA, USA) at a density of 6×10⁵/well and 1×10⁶/well, respectively. Four hours later, the cells were treated with different concentrations of baicalin (10, 30, or 100 µM) dissolved in fresh medium or vehicle, and incubated for another 72 h. Then cells were fixed with 10% formaldehyde solution and stained with 0.25% crystal violet. After being washed with PBS, crystal violet was dissolved and the absorbance was read at 570 nM using a microplate reader (ELx800; BioTek, Winooski, VT, USA).

MDA-MB-231 and 4T1 breast cancer cells were seeded in 6-well plates (Corning, MA, USA) at a density of 2×10⁵/well. Scrapes were made over the cells by a sterile toothpick until cells formed a confluent monolayer. After scraping, the cells were washed with PBS twice, and then replaced with fresh medium with or without various concentrations of baicalin and continued to culture. The width of the wound was monitored and photographed using a microscope (Nikon, Tokyo, Japan) at 100-fold magnification until the wound of the control group healed or almost healed.

The migration assay was performed in 24-well Transwell chambers (Corning, MA, USA). Briefly, complete DMEM with 10% FBS with or without various concentrations of baicalin was added into the lower chambers, and MDA-MB-231 (10⁵) and 4T1 breast cancer (6×10⁴) cells suspended in serum-free DMEM were added into the upper chambers. After incubation for 18 h, the cells were fixed with 10% formaldehyde solution and stained with 0.25% crystal violet. Then the non-migration cells on the upper chamber were wiped off and the migrated cells in the lower chamber were photographed under a microscope (Nikon) at 100-fold magnification.

MDA-MB-231 and 4T1 breast cancer cells were seeded in 24-wells and treated with 100 µM baicalin for 24 h. After being fixed with 4% paraformaldehyde and permeabilized with 0.05% Triton X-100 at room temperature, the cells were incubated with primary antibodies anti-vimentin (1:100) or anti-Slug (1:50) overnight at 4°C. After a further wash with PBS, the cells were incubated with Alexa Fluor 594-conjugated secondary antibody (dilution 1:500) for 1 h. Then, anti-F-actin-Green 488 (dilution 1:500) was added to stain the cytoskeleton and the nuclei were stained by DAPI. Images were captured at ×200 under a fluorescence microscope (Nikon).

Tissues were fixed, dehydrated, embedded and cut into 5-µm sections in accordance with standard procedures. After deparaffinization, rehydration, antigen retrieval and blockage, the sections were incubated with primary antibodies overnight at 4°C. Then the sections were blocked the endogenous peroxidase with hydrogen peroxide and incubated with the biotinylated goat anti-rabbit secondary antibodies (1:100). After signal amplification with avidin and horseradish peroxidase (HRP)-conjugated biotin, the sections were counterstained with DAB to visualize the nuclei. Finally, the sections were mounted and imaged.

Cells were lysed in cell lysis buffer containing various enzyme-protecting agents to collect total proteins. The concentration of protein was determined by the BCA kit. Total proteins (40 µg) were separated on 10% SDS gel and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocked with 5% BSA in Tris-buffered saline (TBS) containing 0.1% Tween-20, the membranes were incubated with the primary monoclonal antibody at 4°C overnight. The membranes were washed with TBST three times, followed by incubation with the secondary antibodies labeled with horseradish peroxidase (HRP). Finally, the membranes were visualized with an enhanced chemiluminescent system.

Total RNA from cancer cells was extracted by using the RNA isolation kit according to the manufacturer's instructions, and cDNA was synthesized using PrimeScript RT reagent kit with 1 µg total RNA. qPCR was performed by using the PCR kit according to the instructions. The following primer sequences were used: E-cadherin sense, 5′-TCCTGGGCAGAGTGAATTTTGAAGA-3′ and antisense, 5′-AAACGGAGGCCTGATGGGG-3′; claudin sense, 5′-CCTCCTGGGAGTGATAGCAAT-3′ and antisense, 5′-GGCAACTAAAATAGCCAGACCT-3′; vimentin sense, 5′-TACAGGAAGCTGCTGGAAGG-3′ and antisense, 5′-ACCAGAGGGAGTGAATCCAG-3′; N-cadherin sense, 5′-AGCCAACCTTAACTGAGGAGT-3′ and antisense, 5′-GGCAAGTTGATTGGAGGGATG-3′; Snail sense, 5′-TCGGAAGCCTAACTACAGCGA-3′ and antisense, 5′-AGATGAGCATTGGCAGCGAG-3′; Slug sense, 5′-GGGGAGAAGCCTTTTTCTTG-3′ and antisense, 5′-TCCTCATGTTTGTGCAGGAG-3′; and GAPDH sense, 5′-TGTTGCCATCAATGACCCCTT-3′ and antisense, 5′-CTCCACGACGTACTCAGCG-3′. Relative quantification was achieved by normalization to GAPDH.

Six- to eight-week-old female BALB/c mice were obtained from the Laboratory Animal Center of Chongqing Medical University and housed in a specific pathogen-free (SPF) laboratory environment. BALB/c mice were injected subcutaneously with 1×10⁶ 4T1 cells into the bilateral gluteal region. One week after inoculation, the mice were divided randomly into a sham-treated group and a baicalin-treated group. The former received PBS while the latter received 100 mg/kg baicalin in an intraperitoneal injection approach every 3 days. After 6 weeks, all mice were sacrificed under anesthesia, the tumors and lungs were excised, weighed, counted for tumor nodules and fixed for further analysis.

All data wre analyzed with SSPS 13.0. Data are expressed as mean ± SD. Variances between groups were analyzed using the Student's t-test and one-way analysis of variance (ANOVA). P<0.05 was considered to indicate a statistically significant result.

Previous studies have revealed that baicalin exerts beneficial effects on inhibiting cell proliferation and inducing apoptosis in various cancer types (24–26). To evaluate whether baicalin affects the viability of breast cancer cells, the crystal violet assay was performed in MDA-MB-231 and 4T1 cells. As shown in Fig. 1B and C, the cell viability had no significant difference when the breast cancer cells were treated with increasing concentrations of baicalin up to 100 µM.

To determine whether baicalin has the potential to inhibit breast cancer cell migration and invasion, wound healing and transwell migration assays were performed using two highly aggressive breast cancer cell lines MDA-MB-231 and 4T1. In the wound healing assay, following pretreatment with baicalin (10, 30, or 100 µM), the wound scratches in the baicalin-treated groups were wider than those noted in the control group at 18 h, and had a dose-dependent increasing trend (Fig. 2A and B), indicating that baicalin inhibited cell migration of breast cancer cells. In the cell invasion experiment by Transwell assay, results similar to those of the wound healing assay were observed in the baicalin-treated breast cancer cells (Fig. 2C and D).

It has been confirmed that EMT plays a major role in tumor metastasis. In order to explore the relationship between baicalin and EMT, we used varying concentrations of baicalin to treat MDA-MB-231 and 4T1 breast cancer cells, respectively. IF assay indicated that the mesenchymal marker vimentin was degraded and the cytoskeletal protein F-actin was remolded after the two breast cancer cell lines were treated with 100 µM baicalin (Fig. 3A), and the expression of Slug, one of the major EMT TFs that is usually maintained at a high level in highly invasive breast cancer cells (27), was downregulated in the baicalin-treated MDA-MB-231 and 4T1 cells (Fig. 3B). Furthermore, western blotting and quantitative RT-PCR also showed that epithelial markers E-cadherin and claudin were upregulated while mesenchymal markers N-cadherin and vimentin and relative TFs Snail and Slug were downregulated in a dose-dependent manner by baicalin in the two highly invasive breast cancer cell lines (Fig. 3C and D).

Prior studies have demonstrated that baicalin can inhibit breast cancer cell migration through the p38 MAPK signaling pathway (18). Cancer metastasis and EMT are complex processes which are influenced by numerous signaling pathways through crosstalk (6). In view of the important status of β-catenin in cancer metastasis and EMT, we first determined whether baicalin affects the β-catenin signaling pathway. Western blotting and quantitative RT-PCR analysis found that β-catenin was markedly expressed in the MDA-MB-231 and 4T1 breast cancer cells, and pretreatment with baicalin dose-dependently downregulated the expression of β-catenin protein and mRNA (Fig. 4A and B). In addition, overexpression of β-catenin in the baicalin-treated 4T1 cells by adenovirus vector system, significantly blunted the role of baicalin in regards to the expression levels of EMT-related molecules and TFs, returning cells back to a mesenchymal type (Fig. 4C and D). Simultaneously, the inhibitory effects of baicalin on the migration and invasion of highly invasive breast cancer cells were also reversed with the overexpression of β-catenin (Fig. 4E and F).

Finally, we constructed a xenograft metastasis tumor model of 4T1 breast cancer cells to investigate the effects of baicalin on breast cancer metastasis in vivo. As shown in Fig. 5A and B, we found that the numbers of metastatic nodules on the surface of the liver and lung in the baicalin-treated group were less than these numbers in the control group. H&E staining of the liver and lung also indicated that baicalin reduced the metastatic lesions of breast cancer cells in the liver and lung tissues (Fig. 5C). Similar to the in vitro experiment, the expression levels of mesenchymal marker vimentin and EMT-activating TF Slug in the orthotopic tumor tissues were downregulated by baicalin (Fig. 5D). Finally, immunohistochemistry showed that baicalin inhibited β-catenin expression in the orthotopic tumor tissues (Fig. 5E).