The human HCC cell line, MHCC97-H, was obtained from the Liver Cancer Institute of Fudan University in Shanghai. The cells were cultured at 37°C in 50 mL/L CO₂ air in high glucose Dulbecco’s Modified Essential Medium (DMEM; Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco BRL, Grand Island, NY, USA). Genistein (5,7,4’-trihydroxyisoflavone) purchased from Sigma Chemical Co. (St. Louis, MO, USA) was suspended in dimethyl sulfoxide (DMSO) for the experiments.

A 96-well plate was incubated with exponentially growing cells at a density of 1 × 10⁴ per well. Following incubation of MHCC97-H cells with or without genistein in different columns of 96-well microtiter plates, on day 1-6 methyl thiazol terazolium (MTT) was added to each well and incubated at 37°C for another 4 h before spectrophotometric detection (A₅₉₅). Each assay was performed in quadruplicate.

The inhibitory rate of tumor cell growth was calculated as: (average A₅₉₅ value of control group-average A₅₉₅ value of genistein group)/average A₅₉₅ value of control group[4].

Pre-coating of 96-well microtiter plates was performed by incubating wells with 20 mg/L fibronectin at 4°C overnight. The wells were then blocked with 2% bovine serum albumin (BSA) for 45 min at 37°C. Cells were cultured in medium (DMEM with 2% FBS) for 24 h, then harvested at about 70% confluence, resuspended in serum-free DMEM medium supplemented with 0.1% BSA and distributed in the wells (8 × 10⁴ cells/well). The cells were incubated at 37°C in a 50 mL/L CO₂ atmosphere for 20, 40, 60 and 90 min with or without genistein. The wells were washed 3 times with phosphate buffered saline (PBS) to remove unattached cells, and the attached cells were then incubated with MTT and the A₅₉₅ was measured.

The invasive activity of MHCC97-H cells was assayed in Transwell cell chambers (Corning Inc., Corning, NY, USA), according to the method reported by Kido et al[5]. Polyvinylpyrrolidone-free polycarbonate filters with an 8.0 μm pore size were pre-coated with 5 μg of fibronectin in a volume of 50 μL on the lower surface. The Matrigel was diluted to 100 μg/mL with cold PBS, applied to the upper surface of the filters (5 μg/filter), and dried overnight under a hood at room temperature. Log-phase cell cultures of MHCC97-H cells were harvested and washed 3 times with serum-free DMEM, then resuspended at a final concentration of 2 × 10⁶ cells/mL in DMEM with 0.1% BSA. Cell suspensions (100 μL) with or without genistein were added to the upper compartment and incubated for 20 h at 37°C in a 50 mL/L CO₂ atmosphere. The filters were fixed with methanol and stained with Giemsa. The cells on the upper surface of the filters were removed by wiping with cotton swabs. The cells invading the lower surface of the filter through the Matrigel and the filter were manually counted under a microscope at a magnification of × 400. Each assay was performed in triplicate. The inhibitory rate of adhesion and invasion were calculated[4].

MHCC97-H cells were seeded at a density of 5 × 10⁵ cells/well in six-well dishes. After 24 h the cells were treated with or without genistein for 72 h and harvested by trypsinization. The cells were then centrifuged at 300 g for 10 min, washed in PBS, and resuspended in cold 70% ethanol. The cells were then subjected to flow cytometric analysis on a FACScan cytofluorimeter (Becton Dickinson, Franklin Lakes, NJ, USA) after propidium iodide labeling.

Control and genistein-treated cell extracts were prepared and the proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by Western blot analysis using a FAK monoclonal antibody (1:1000; SC-713; Cell signaling, USA) and pFAK polyclonal antibody (1:500; SC-6243; Cell signaling, USA) as primary antibodies. Anti-rabbit/mouse immunoglobulin G (IgG)-HRP (Beyotime Biotech, China) was used as a secondary antibody, followed by the detection of chemiluminescence using chemiluminescence kits (Beyotime Biotech, China). The optical density was determined using a scanning densitometer and analyzed using Quantity One software (Bio-Rad). House-keeping gene β-actin was used as an internal standard.

Male athymic BALB/c nu/nu mice (46-wk-old), were obtained from the Shanghai Institute of Materia Medica, Chinese Academy of Science and maintained in specific pathogen-free (SPF) conditions and fed with sterilized MF pellets and distilled water. All studies on the mice were conducted in accordance with the National Institutes of Health “Guide for the Care and Use of Laboratory Animals”. The mouse study protocol was approved by the Shanghai Medical Experimental Animal Care Commission.

MHCC97-H cells (5 × 10⁶) in 0.2 mL of serum-free culture medium were injected subcutaneously into the upper flank region of nude mice, and the mice were observed for tumor growth. When a subcutaneous tumor had reached approximately 1.5 cm in diameter, a small piece was removed and cut into pieces of approximately 1 mm × 1 mm × 1 mm which were subsequently implanted into the livers of 12 new recipient nude mice by a method described previously[6]. Of the 12 recipient nude mice, bearing an orthotopic tumor implant, 6 were randomly selected for treatment with genistein and the remaining 6, which did not receive genistein treatment, served as controls. Genistein (50 mg/kg) was administered intraperitoneally to each mouse in the genistein group daily for 20 d, while control animals were administered the same vehicle. The mice were observed for 35 d, and then killed by cervical dislocation. The liver and lungs were excised during autopsy, lungs were fixed and embedded in paraffin and coronal sections were cut. After hematoxylin and eosin staining, micrometastatic foci were counted microscopically.

All data are the mean ± SD. The statistical significance of differences between the treated and control groups were determined by applying the one-way ANOVA and χ² test. The statistical analysis software package Stata 6.0 was used for the tests, and P < 0.05 was considered statistically significant.

Genistein significantly inhibited MHCC97-H cell growth over the 6-d experimental period. The inhibitory rate of tumor cell growth in the 5, 10 and 20 μg/mL genistein groups was 22.3%, 58.2%, and 80.1%, respectively. The inhibitory rate of MHCC97-H cell growth in the 10 and 20 μg/mL genistein groups was significantly higher than the 5 μg/mL genistein group (P < 0.05). The concentration-dependent effects of genistein on MHCC97-H cell growth are shown in Figure 1.

The A₅₉₅ for MHCC97-H cells treated with or without genistein in fibronectin pre-coated 96-well microtiter plates at 20, 40, 60 and 90 min is shown in Table 1. Our results showed that genistein significantly inhibited tumor cell adhesion to fibronectin-coated substrates in a concentration-dependent fashion (P < 0.05), and the inhibitory effect of genistein on adhesion was more potent in the 10 and 20 μg/mL genistein groups. The inhibitory rate of genistein on MHCC97-H cell adhesion is shown in Figure 2.

We also investigated the capability of metastatic tumor cells to migrate through reconstituted basement membrane (Matrigel). The cells invading the lower surface of the filter through Matrigel in the control group, 5, 10, and 20 μg/mL genistein groups were 234.20 ± 12.36/field, 213.60 ± 14.98/field, 193.80 ± 19.92/field, and 142.80 ± 23.66/field, respectively. The inhibitory rate of invasion is shown in Figure 2. Our results showed that genistein inhibited the in vitro invasion of MHCC97-H cells. The inhibitory effect on invasion of MHCC97-H cells in the 20 μg/mL genistein group was more significant than that in the 5 and 10 μg/mL genistein groups (P < 0.05).

The effects of genistein on the cell cycle in MHCC97-H cells were determined by flow cytometry. After treatment with genistein, the number of MHCC97-H cells in the G₀/G₁ phase increased significantly compared with control cells (P < 0.05). The number of cells in the G₂/M phase was decreased, but there was no statistical significance between the control group and the 10 and 20 μg/mL genistein-treated groups (P > 0.05). S phase fractions decreased significantly in cells treated with 10 and 20 μg/mL genistein (P < 0.01). The percentage of apoptotic cells in the genistein-treated groups increased significantly compared with the control group (P < 0.05). The results of the cell cycle analysis of MHCC97-H cells by flow cytometry are presented in Table 2.

The effect of genistein on expression and phosphorylation of FAK in MHCC97-H cells was determined using Western blotting. After treatment with genistein, the expression level of total FAK was decreased when the genistein concentration increased (P < 0.05). The optical density in the 10 μg/mL and 20 μg/mL genistein groups were significantly lower than that in the control group (P < 0.05). More interestingly, at the same time phosphorylated FAK also decreased. The 5 μg/mL genistein treatment inhibited about 40% of FAK phosphorylation. These parameters were further significantly inhibited in the presence of genistein in a concentration-dependent fashion (P < 0.05). Data are shown in Figures 3 and 4.

The anti-tumor activity of genistein was evaluated in nude mice bearing orthotopic tumor implants

(Figure 5A). Treatment with genistein significantly inhibited local tumor growth, compared with the control group. At the end of treatment, the tumor weight in the genistein group was significantly less than that in the control group (1.99 ± 2.07 g vs 2.63 ± 1.31 g, P < 0.05). Tumors in mice treated with genistein were reduced in weight by 24.3% compared with the control group.

After orthotopic implantation of tumor tissues, pulmonary metastases occurred in the recipient mice by day 35 (Figure 5B). Microscopy showed that the number of micrometastatic foci in the genistein group (12.3 ± 1.8) was significantly lower than that in the control group (12.3 ± 1.8 vs 16.6 ± 2.6, P < 0.05).

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer related deaths in the world. A high incidence of metastasis accounts for the poor survival of HCC patients, and research into interventions for liver cancer metastasis has special priority in the anti-cancer campaign.

Genistein, an isoflavonoid abundant in soy beans, has a wide range of biological actions in the inhibition of tumors including breast cancer, prostate cancer, colon cancer, leukemia and melanoma. However, few data are available on the effects of genistein on metastasis of HCC. In this study, the authors demonstrate that genistein inhibited the metastasis of HCC in vitro and in vivo. Genistein appears to be a promising agent for inhibiting the metastatic potential of HCC.

In this study, genistein was found to significantly inhibit the growth, attachment and invasion of MHCC97-H cells in vitro in a concentration-dependent fashion. The expression and phosphorylation of FAK in MHCC-97H cells were also decreased by genistein. In addition, our study on nude mice bearing orthotopic tumor implants showed that genistein significantly inhibited lung metastasis of MHCC97-H cells.

The findings from this study support the idea that genistein may serve as a potentially important anticancer agent for HCC progression by blocking the FAK signaling process.

Genistein is a planar molecule with an aromatic A ring, has a second oxygen atom from that in the A ring, and has a molecular mass similar to those of the steroidal estrogens. It binds to and inhibits protein tyrosine kinase, thereby disrupting signal transduction and inducing cell apoptosis and differentiation.

This paper reports the effect of genistein exposure on the growth, adhesion, migration and metastases of a highly metastatic HCC cell line MHCC97-H. They observed that genistein exposure significantly inhibited growth, adhesion to fibronectin, and invasion of the cell line. The paper is well written and has novel data.