Baicalein inhibits the invasion and metastatic capabilities of hepatocellular carcinoma cells via down-regulation of the ERK pathway

Abstract

Baicalein, a widely used Chinese herbal medicine, has historically been used in anti-inflammatory and anti-cancer therapies. However, the anti-metastatic effect and molecular mechanism(s) of baicalein on hepatocellular carcinoma (HCC) remain poorly understood. Therefore, the purpose of this study was to assess the anti-metastatic effects of baicalein and related mechanism(s) on HCC. Based on assays utilized in both HCC cell lines and in an animal model, we found that baicalein inhibited tumor cell metastasis in vivo and in vitro. Furthermore, after treatment with baicalein for 24 hours, there was a decrease in the levels of matrix metalloproteinase-2 (MMP-2), MMP-9 and urokinase-type plasminogen activator (u-PA) expression as well as proteinase activity in hepatocellular carcinoma MHCC97H cells. Meanwhile, the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 were increased in a dose-dependent fashion. Moreover, baicalein treatment dramatically decreased the levels of the phosphorylated forms of MEK1 and ERK1/2. MEK1 overexpression partially blocked the anti-metastatic effects of baicalein. Combined treatment with an ERK inhibitor (U0126) and baicalein resulted in a synergistic reduction in MMP-2, MMP-9 and u-PA expression and an increase in TIMP-1 and TIMP-2 expression; the invasive capabilities of MHCC97H cells were also inhibited. In conclusion, baicalein inhibits tumor cell invasion and metastasis by reducing cell motility and migration via the suppression of the ERK pathway, suggesting that baicalein is a potential therapeutic agent for HCC.

Figure 1. Chemical structure of baicalein.

Figure 2. Baicalein inhibits the proliferation of MHCC97H cells.

Cell viability was measured by MTT assay. Values represent the means ± standard deviation (SD) of three independent experiments performed in triplicate. *p < 0.05 and **p < 0.01 compared with the control group.

Figure 3. Baicalein inhibits the motility and invasiveness of MHCC97H cells.

(A) MHCC97H cells were wounded and then incubated in media containing 2% serum with varying concentrations of baicalein (0, 10, 20 and 30 µM) for 24 h. Pictures were taken at 0, 6, 12, 18 and 24 h after addition of baicalein. (B) The percent migration rate is expressed as a percentage of the control (0 µM). (C) MHCC97H cells were pretreated with 0, 10, 20 and 30 µM baicalein for 24 h and were then seeded in the upper wells. FBS (10%) was added to the bottom chambers for 24 h to induce cell invasion. After 24 h, cells on the bottom side of the filter were fixed, stained and counted. (D) The percent invasion rate was expressed as a percentage of the control (0 µM). Values represent the means ± SD of three independent experiments performed in triplicate. *p < 0.05 and **p < 0.01 compared with the control group.

Figure 4. Baicalein suppresses the expression and activity of MMP-2, MMP-9 and u-PA and promotes the expression of TIMP-1 and TIMP-2 in MHCC97H cells.

(A) The effects of baicalein on the expression levels of MMP-2, MMP-9 and u-PA mRNA were assessed by qRT-PCR. (B) MHCC97H cells were treated with baicalein (0, 10, 20 and 30 µM) for 24 h and then subjected to western blotting to analyze the protein levels of MMP-2, MMP-9 and u-PA. (C) Quantification of the protein levels of MMP-2, MMP-9 and u-PA. (D) Effects of baicalein on the activities of MMP-2, MMP-9 and u-PA. (E) Quantification of the activities of MMP-2, MMP-9 and u-PA. (F) MHCC97H cells were treated with baicalein (0, 10, 20 and 30 µM) for 24 h and then subjected to western blotting to analyze the protein levels of TIMP-1 and TIMP-2. (G) Quantification of the protein levels of TIMP-1 and TIMP-2. Values represent the means ± SD of three independent experiments performed in triplicate. *p < 0.05 and **p < 0.01 compared with the control group.

Figure 5. Effect of baicalein on the ERK pathway.

(A) The protein levels of MEK1, p-MEK1, ERK1/2 and p-ERK1/2. (B) Phosphorylation densities of MEK1 and ERK1/2 were digitally scanned. (C) Transwell assays were performed to evaluate the anti-metastatic effects of baicalein on two groups of cells transfected with the indicated plasmids (transfected with the empty vector pcDNA3.1 (+) or with pcDNA3.1 (+)-MEK1, ‘pcDNA3.1(+)’ means the group transfected with an empty vector pcDNA3.1(+), ‘pcDNA3.1(+)-MEK1’ means the group transfected with a pcDNA3.1(+)-MEK1). (D) The percent invasion rate was expressed as a percentage of the control (the control group of cells transfected with an empty vector pcDNA3.1 (+)). (E) The inhibition rates of baicalein on two groups of cells. Values represent the means ± SD of three independent experiments performed in triplicate. *p < 0.05 and **p < 0.01 compared with the control group.

Figure 6. Effects of the ERK inhibitor (U0126) and baicalein on cell invasion and MMP-2, MMP-9, u-PA, TIMP-1 and TIMP-2 expression in MHCC97H cells.

(A) Cells were pretreated with U0126 (10 μM) for 30 min and then incubated in the presence or absence of baicalein (10 μM) for 24 h. Cellular invasiveness was measured using the Boyden chamber invasion assay. (B) The percent invasion rate was expressed as a percentage of control. (C, D) MHCC97H cells were treated and then subjected to western blotting to analyze the protein levels of MMP-2, MMP-9, u-PA, TIMP-1 and TIMP-2. Values represent the means ± SD of three independent experiments performed in triplicate. *p < 0.05 and **p < 0.01 compared with the control group.

Figure 7. The anti-metastastic effects of baicalein on HCC in vivo.

Microscopic findings of lung metastasis in the control group (A) and the treated group (B); black arrows show lung metastasis (100x). (C) The lung metastatic rate of the control and treated group. (D) The average number of lung metastasis in the control and treated group. *p < 0.05 and **p < 0.01 compared with the control group.