CRC HT29 and DLD1 cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in RPMI-1640 (HyClone Laboratories, Inc., Logan, UT, USA) medium with 10% fetal bovine serum (FBS; HyClone Laboratories). The cells were cultured at 37°C with 5% CO₂. Anti-MMP-2, ERK, MMP-9 and p-ERK were purchased from Cell Signaling Technology (Beverly, MA, USA). Unless otherwise indicated, all the other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Apoptotic and/or necrotic cells were analysed by propidium iodide (PI) uptake using Annexin V binding an Annexin V-FITC/PI kit as previously described (15). Briefly, CRC cells were seeded into 6-well plates at a density of 1×10⁵ cells/well for 12 h and treated with 0, 40, 60 and 80 µM of baicalein for 48 h. After washing with cold phosphate-buffered saline (PBS), cells were resuspended in Annexin V binding buffer. Cells were incubated in Annexin V-FITC for 15 min, washed and then incubated with PI. Samples were detected by flow cytometer with CellQuest software.

Cell growth rate was detected using the MTT assay. Briefly, cells were seeded in a 96-well plate at a density of 1.5×10³ cells. Cells were treated with baicalein (0, 10, 20, 30, 40, 50, 60, 80, 100 and 120 µM) (R&D Systems, Inc., Minneapolis, MN, USA) for 24 h. MTT assay was done in accordance with the manufacturers instructions. Each experiment was done in triplicate and data were expressed as mean ± SD. In focus formation assay, 100 cells were firstly seeded onto a plate and then cultured with (0, 40, 60 and 80 µM) baicalein for 2 weeks. The plates were fixed and then stained with 1% crystal violet. All experiments were performed in triplicate.

The construction of the expression plasmids and their transfection was performed in accordance with the manufacturers instructions (3). First, the full-length pcDNA3.1 (Invitrogen, Carlsbad, CA, USA) MEK1 vector was made by cloning the full-length PCR product of MEK1 with KOD^® DNA polymerase (Toyobo Co., Ltd., Osaka, Japan). Then we measured the plasmid sequences to confirm the sequences of the plasmid. In transient transfection experiments, cells were plated at a density of 2×10⁵ cells/well in a 6-well plate, 24 h later they were transfected with 4.0 µg pcDNA3.1(+)-MEK1 vector via Lipofectamine 2000 (Invitrogen) in accordance with the manufacturers protocol. The 4.0 µg pcDNA3.1(+) empty vector was used as a negative control.

The migration and invasion ability of cells were detected by Transwell assays. Migration and invasion assay was performed with 24-well Transwell chambers coated with Matrigel or not (Becton-Dicknson, Billerica, MA, USA) as previously described. For the migration assay, cells were seeded into inner well and cultured in medium with 0, 10, 20 and 30 µM baicalein. After 16 h, cells on the bottom side of the inner well were fixed in alcohol, fixed in crystal violet and counted. Invasion assay was performed with 24-well Transwell chamber coated with Matrigel as previously described (3).

After treated with U0126 or baicalein, 2×10⁶ cells were added into 200 µl of lysis buffer (Fermentas, Waltham, MA, USA). By 10% SDS-polyacrylamide gel electrophoresis, the proteins (60 µg) were separated and bloted onto PVDF membranes. To block non-specific binding, the PVDF membranes were subsequently blocked in Tris-buffered saline with Tween-20 (TBST) containing 5% defatted milk buffer for 1 h at 37°C and were then incubated with antibodies against ERK, p-ERK, MMP-2, MMP-9 or β-actin overnight in defatted milk 5% in TBST at 4°C. The membranes were incubated with a horseradish peroxidase goat anti-rabbit or anti-mouse IgG antibody at room temperature for 1 h. The pictures were examined with an enhanced chemiluminescence kit (ECL Plus; Amersham, Freiburg, Germany) according to the manufacturers instructions and captured by autoradiography. The relative photographic density was quantified with ImageJ software (GE Healthcare, Buckinghamshire, UK) and expressed as arbitrary units (a.u.).

To assess the anti-proliferation of baicalein in vivo, Balb/c athymic nude mice (4- to 6-week-old) were obtained from Shanghai SLAC Laboratory Animal, Co., Ltd., (Shanghai, China). Each mouse was subcutenously implanted with 1×10⁶ viable DLD1 cells in the right flank area. One group was treated with vehicle dimethyl sulfoxide (DMSO), the other group was treated with baicalein (20 mg/kg/day) for 3 weeks. At day 21, all the mice were sacrificed and the tumor tissues were removed and weighed. The experimental protocols used herein were evaluated and approved by the Animal Care and Use Committee of the Medical School of Xian Jiaotong University.

The inhibition effects of baicalein on the proliferation of CRC cells at different concentrations (0 to 120 µM) for 24 h are shown in Fig. 1B. At concentrations >40 µM, the anti-proliferation effect of baicalein on HT29 and DLD1 cells was significant so we chose concentrations <40 µM for all the following experiments.

To determine the effect of baicalein on apoptosis in detail, HT29 cells were incubated for 48 h with baicalein (40, 60 or 80 µM) and then analysed by flow cytometry. As shown in Fig. 1C, the effect of baicalein on apoptosis of HT29 cells was concentration-dependent. After treatment for 48 h, both early and late apoptotic cells increased significantly in the HT29 cells treated with baicalein. Baicalein reduced the number of colony HT29 cells in vitro significantly (Fig. 1D).

Fig. 2 shows the inhibition effect of baicalein (0, 10, 20 and 30 µM) on cell migration (16 h) and invasion (24 h) in HT29 and DLD1 cells, respectively. We found that inhibition effect of baicalein on the migration (Fig. 2A and B) and invasion (Fig. 2C and D) of HT29 and DLD1 cells was concentration-dependent.

We analysed the effect of baicalein on the expression of MMP-2/−9 in HT29 and DLD1 at various concentrations. Cells were treated with baicalein (0, 10, 20 and 30 µM) for 24 h and then analysed by western blotting. Fig. 3A and B show the inhibited effect of baicalein on the expression of MMP-2/−9 in HT-29 cells. The effect is concentration-dependent. Fig. 3C and D show the effect of baicalein on the expression of MMP-2/−9 in DLD1 cells.

ERK signaling pathway plays an vital role in the metastasis of cancer cells by regulating MMP-2/−9 (3); hence, we detected the impact of baicalein on the activity of ERK signaling pathway in CRC cells. The results of the western blotting showed that baicalein reduced the phosphorylation level of ERK1/2 in DLD1 cells treated with baicalein (Fig. 4A and B). To further check the role of ERK signaling pathway in the anti-metastatic effect of baicalein, we upregulated the activity of ERK singnaling pathway in DLD1 cells via transfecting the plasmid [pcDNA3.1 (+)-MEK1] expressing human MEK1 and found that the anti-invasion effect of baicalein was reversed by the high expression of MEK1 (Fig. 4C). The positive clones were selected by G418. DLD1 cells were separated into 3 groups: no treatment as the control group ‘C’, transfected with an empty vector pcDNA3.1(+) as the negative control group ‘N’, transfected with a pcDNA3.1(+)-MEK1 as the positive group ‘M’.

The inhibition rate was ~61.3 and 39.3% after 24 h of treatment with 0 or 30 µM of baicalein (Fig. 4D). Baicalein could also inhibit the phosphorylation level of ERK in the HT29 cells (data not shown). Our results also show that the combined treatment with the baicalein and U0126 (an ERK inhibitor) reduced both the MMP-2/−9 protein expression (Fig. 5A and B) and the cell invasion significantly (Fig. 5C and D).

The inhibition effect of baicalein on DLD1 xenograft growth is shown in Fig. 6A. We found that baicalein could inhibit CRC growth significantly. At day 21, all the mice were sacrificed and the tumor tissues were removed and weighed. Compared to the control group, baicalein reduced the volume of solid tumors significantly (Fig. 6B). We also found that baicalein significantly inhibited phosphorylation level of ERK and the expression of MMP-2/−9 in vivo. The inhibition effect of baicalein on the phosphorylation level of ERK is shown in Fig. 6C and D, and on expression of MMP-2/−9 is shown Fig. 6E and F in vivo.