The human HCC cell line, Bel 7402, was obtained from Cancer Institute of Sun Yat-Sen University in Guangzhou. The cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) and cultured at 37 °C in a humidified atmosphere containing 50 mL/L CO₂ in air. Genistein purchased from Sigma Chemical Co. was suspended in dimethylsulfoxide (DMSO) for the experiments.

The cells were seeded at the density of 1×10⁴ cells with 1mL of medium/well onto 24 plates and incubated with or without genistein for 6 d. On the indicated day thereafter, cells were trypsinized and the number of cells was scored. An equivalent volume of DMSO was added to control cultures.

Cell viability was assayed using methyl thiazol tetrazolium (MTT) method. A 96-well plate was incubated with exponentially growing cells at the density of 1×10⁴/well, following incubation of Bel 7402 cells with or without genistein in different columns of 96-well microtiter plates on d 1, 3, 5, and 7, MTT was added to each well and incubated at 37 °C for further 4 h before 595 nm absorbance (A_595nm) was detected. Each assay was performed in quadruplicate.

Inhibitory rate of tumor cell growth = (average A_{595 nm} value of control group-average A_{595 nm} value of genistein group)/average A_{595 nm} value of control group[12].

Ninety-six-well microtiter plates were precoated with 20 mg/L fibronectin and incubated at 4 °C overnight. Wells were blocked with 2% BSA for 45 min at 37 °C. The cells were cultured in 2% serum-containing medium for 24 h, harvested at about 70% confluency, resuspended in serum-free RPMI 1640 medium supplemented with 0.1% BSA and distributed to wells (8×10⁴/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 thrice with PBS to remove unattached cells, then the attached cells were incubated with MTT and the absorbance was measured at 595 nm. Each assay was performed in triplicate.

Adhesion rate = average A_{595 nm} value of genistein group/average A_{595 nm} value of control group×100%.

Inhibitory rate of adhesion = (average A_{595 nm} value of control group-average A595 nm value of genistein group)/average A595 nm value of control group×100%.

The invasive activity of Bel 7402 cells was assayed in transwell cell chambers (Corning Inc., USA), according to the method reported by Kido et al[13]. The basement membrane Matrigel was obtained from the Department of Cell Biology, Peking University Health Science Center. Polyvinylpyrrolidone-free polycarbonate filters with an 8.0-μm pore size were precoated 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 and applied to the upper surface of the filters (5 μg/filter), and dried overnight under a hood at room temperature. The coated filters were washed extensively in PBS, and then dried immediately before use. Log-phase cell cultures of Bel 7402 cells were harvested and washed thrice with serum-free RPMI 1640, and resuspended to a final concentration of 2×10⁶/mL in RPMI 1640 with 0.1% BSA. Cell suspensions (100 μL) with or without genistein were added to the upper compartment and incubated for 10-20 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 Matrigel and filter were manually counted under a microscope at a magnification of ×400, and each assay was performed in triplicate.

Invasion rate = average cell numbers invading the lower surface of the filter in genistein group/average cell numbers invading the lower surface of the filter in control group×100%.

Inhibitory rate of invasion=(average cell numbers invading the lower surface of the filter in control group-average cell numbers invading the lower surface of the filter in genistein group)/average cell numbers invading the lower surface of the filter in control group×100%.

Bel 7402 cells were seeded at the density of 5×10⁵/well in six-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 r/min 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) after propidium iodide labeling.

On d 3 of cell culture, control and genistein-treated Bel 7402 cells were centrifuged at 300 r/min for 10 min, washed and fixed in 1 mL 70% ethanol at 4 °C, treated with 0.1% Triton X-100 and 1% FBS, and anti-focal adhesion kinase (FAK C-20 sc-558, Santa Cruz Biotechnology, Inc., USA) and IgG1 (1∶100) were added. Cells were then treated with RNAse (1 mg/mL, Sigma, USA) and propidium iodide (10 μg/mL) for 30 min at room temperature. FAK expression was measured using a FACScan cytofluorimeter (Becton Dickinson).

Control cells and genistein-treated cells were harvested at 72 h by trypsinization and collected by centrifugation. Cells were washed twice in PBS-0.1% bovine serum albumin and prepared for TUNEL assay. Cells were fixed for 30 min in 4% paraformaldehyde, washed twice in PBS, and then permeabilized in 0.1% Triton-X 100 and 0.1% sodium citrate. Cells were labeled with terminal deoxynucleotidyl transferase for 60 min at 37 °C in a dark humidified incubator. The samples were washed twice, resuspended in 500 μL of PBS, and then analyzed on a FACScan cytofluorimeter (Becton Dickinson).

Six-week-old male BALB/C nu/nu mice were obtained from the Medical Laboratory Animal Center, Sun Yat-Sen University, China. They were kept under sterile conditions in autoclaved cages with filter bonnets in laminar flow units and fed with sterilized MF pellets and distilled water. The mice were maintained in accordance with institutional accredited guidelines.

Bel 7402 cells were grown to 80-90% of confluence and detached with 0.25% trypsin. The cells were washed twice, counted, and resuspended in PBS at 1×10⁷/mL (viability over 95%). Cells (0.3 mL) were injected into the male nude mice. Each animal received two injections, one on each side of the neck. Animals were killed 3 wk after tumor inoculation, when the largest tumors reached about 10 mm in diameter. Surgical excision of primary tumor was carried out, and tumor tissues were cut into 1-mm³ pieces for bilateral subrenal capsule xenograft transplantation in next 10 nude mice anesthetized with chloral hydrate. The xenograft volume was assessed and calculated by the formula: length×width×depth×0.5236.

Mice bearing subrenal capsule xenograft transplant were randomly selected for the treatment with genistein (n  =  5) and those without genistein treatment served as control (n  =  5). Genistein (50 mg/kg) was administered ip daily to each mouse in genistein group for 15 d, while control animals were given the same vehicle. The animals were killed by cervical dislocation 16 d after transplantation of the tissues. Autopsies were performed and the kidney was excised, fixed, and embedded in paraffin, and examined histologically. The inhibitory rate of transplant growth was calculated by comparing the changes in xenograft transplant volume[15,16]. The criteria for invasive capacity of tumor cells in subrenal capsule xenograft transplant were as previously described[14,16,17].

After deparaffinization, dehydration, and washing, sections from xenograft transplant were incubated with trypsin at 37 °C for 30 min, quenched with 0.3% H₂O₂-methanol for 30 min, and blocked with 10% normal goat serum in a buffer containing 100 mL of PBS, 1.0 g of BSA, and 0.1 mL of Tween-20. The sections were treated with a rabbit polyclonal antibody against human factor VIII-related antigen at a 1∶100 dilution with the PBS-BSA-Tween-20 buffer, followed by a biotinylated universal antibody at a 1∶100 dilution. The sections were then treated with avidin-biotin complex followed by 3,3-diaminobenzidine as a substrate for staining. Positive unit (PU) of the microvessels with staining was tested and calculated according to the method described by Shen[17].

The statistical significance of difference between the groups was determined by applying the one-way ANOVA and χ² using the Stata 6.0 program.

Genistein significantly inhibited Bel 7402 cell growth over the 7 d of experiment. The inhibitory rate of tumor cell growth in 5 and 10 μg/mL genistein groups was 26.71%  ± 10.2% and 42.64 ± 16.1%, respectively. The inhibitory rate of tumor cell growth in 10 μg/mL genistein group was significantly higher than that in 5 μg/mL genistein group (P<0.01). The dose-dependent effects of genistein on Bel 7402 cell growth are presented in Figure 1.

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Figure 1 Effects of genistein on Bel 7402 cell proliferation In vitro effects of genistein on adhesion and invasion of Bel 7402 cells.

The adhesion rate of Bel 7402 cells for 20, 40, 60, and 90 min was 30.61%, 56.48%, 61.89% and 81.55% in 5 μg/mL genistein group, and 17.78%, 15.82%, 42.98% and 64.48% in 10 μg/mL genistein group. The inhibitory rate of Bel 7402 cells for 20, 40, 60, and 90 min was 69.39%, 43.52%, 38.11%, and 18.45% in 5 μg/mL genistein group, and 82.22%, 84.18%, 57.02%, and 35.52% in 10 μg/mL genistein group. Our results showed that genistein could inhibit tumor cell adhesion to fibronectin-coated substrates in a concentration-dependent fashion, and more potent inhibitory effect of genistein on adhesion occurred within 40 min.

We also investigated the capability of metastatic tumor cells through reconstituted basement membrane Matrigel. The cells invading the lower surface of the filter through Matrigel in control group, 5 μg/mL genistein group, and 10 μg/mL genistein group were 243.7±12.6/filed, 216.7±21.3/filed, and 174.5±9.6/filed, respectively. The invasion rate in 5 and 10 μg/mL genistein group was 88% and 71%, respectively, the inhibitory rate of invasion was 11% and 28%, respectively. Our results showed that genistein could inhibit the in vitro invasion of Bel 7402 cells, the inhibitory effect on invasion of Bel 7402 cells in 10 μg/mL genistein group was more significant than that in 5 μg/mL genistein group (P<0.05).

Bel 7402 cells treated with genistein in the G0/G1 and G2/M phases increased significantly than cells in control group, the increase of cells in G0/G1 and G2/M phases was more remarkable in 10 μg/mL genistein group than in 5 μg/mL genistein group (P<0.05). S fractions decreased significantly in cells treated with genistein (P<0.05). Percentage of apoptotic cells in genistein-treated group increased significantly compared to that in control group (P<0.05). The results of cell cycle analysis by flow cytometry in Bel 7402 cells are presented in Table 1.

TUNEL assay in our studies showed that the percentage of cells undergoing apoptosis was significantly higher in 10 μg/mL genistein group (1.05 ± 0.09%) and 5 μg/mL genistein group (0.80 ± 0.12%) than in control group (0.43 ± 0.08%, P < 0.01 and P < 0.05, respectively) being consistent with genistein’s ability to induce apoptosis observed in cell cycle analysis.

We investigated whether genistein could modulate protein expression of the signal transduction molecule-p125FAK. After 72-h treatment with genistein, p125FAK expression in 10 μg/mL genistein group (12.89 ± 0.36)% was significantly lower than that in control group (19.75% ± 1.12%, P < 0.05). p125FAK expression in 5 μg/mL genistein group (15.26 ± 0.16)% also decreased, but there was no statistically significant difference between 5 μg/mL genistein group and control group (P > 0.05).

The anti-tumor activity of genistein was evaluated in nude mice bearing subrenal capsule xenograft transplant. Treatment with genistein inhibited the local tumor growth significantly compared to the control group. At the end of the treatment (15 d post implantation), the increase of tumor volume in genistein group (63.32±8.96 mm³) was significantly less than that in control group (79.25±6.85 mm³, P<0.05). Tumors in mice treated with genistein reduced in volume by 20% compared to the control group.

The criteria for invasive capacity of tumor cells in subrenal capsule xenograft transplant were as previously described[17]. The 0-IV invasion rank to renal parenchyma was recorded[14,16]. In the control group, invasion rank 0 was observed in 1 of 10 mice (1/10), invasion rank I in 8 of 10 mice (8/10), and invasion rank II in 1 of 10 mice (1/10). In genistein-treated group, invasion rank 0 was observed in 8/10, rank I in 2/10 and rank II in 0/10 mice. No rank III or rank IV invasion was observed in both groups. Our results showed that treatment with genistein could significantly inhibit the invasion of Bel 7402 cells to the renal parenchyma (P<0.05). Hematoxylin-eosin stained specimens are shown in Figure 2.

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Figure 2 Tumor invasion of renal parenchyma in control (A) and genistein-treated nude mice (B). HE, magnification ×200.

In untreated tumor tissues, tumor cells were arranged in large nests with plenty of blood sinusoids. Whereas, in genistein-treated tumor tissue, tumor cells were characterized by small cancerous nests with scanty blood vessels. PU value of microvessels in the subrenal capsule xenograft transplant, as a marker of tumor angiogenesis, significantly decreased in genistein group (10.422±0.807) compared to that in control group (22.330±5.696, P<0.01). Immunohistochemical staining for the determination of angiogenesis in subrenal capsule xenograft transplant of the nude mice is shown in Figure 3.

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Figure 3 Tumor tissue with plenty of blood vessels (A) and scanty blood vessels (B) in control and genistein-treated nude mice. HE, magnification ×200.