The anti-adhesion trimeric β peptide (DLYYLMDLSYSMKGGDLYYLMDLSYSMKGGDLYYLMDLSYSMK, β3) was expressed in E. coli as described previously (18). Briefly, the DNA fragment of β3 (Sangon Biotech Co., Ltd., Shanghai, China) was cloned into the pET-His expression vector (Gene Power Lab Ltd., Shenzhen, China) and the fusion protein His-β3 was expressed in E. coli BL21 (DE3) plysS (Gene Power Lab Ltd.). Following 1.5 h of induction with isopropyl-β-D-thiogalactoside (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA), 20 mg of β3 peptide was obtained from 1 L of culture medium following purification with metal-chelating sepharose 6B FF (Vector Gene Technology Company Ltd., Beijing, China). The purity of β3 was 92.2% according to Gel-Pro Analyzer 3.1 software (Media Cybernetics, Inc., Rockville, MD, USA).

The highly metastatic HCC cell line HCCLM6, was initially established and preserved by the Liver Cancer Institute, Fudan University (Shanghai, China) and was cultured in Gibco Dulbecco's modified eagle's medium (DMEM, Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Inc.), 100 U/ml penicillin at 37°C under an atmosphere of 5% CO₂. The medium was replenished every 3 days to maintain cell growth.

FN (Sigma-Aldrich, St. Louis, MO, USA) solution (containing 10 µg/ml FN, 20 mmol/l Tris-Cl, pH 7.4, 150 mmol/l NaCl, 1 mmol/l MgCl₂, 1 mmol/l CaCl₂, 1 mmol/l MnCl₂) was added to a 96-well high binding microplate (100 ml per well), and incubated at 4°C overnight. The plate was then incubated with blocking buffer (10 mmol/l Hepes, pH 7.4, 140 mmol/l NaCl, 5.4 mmol/l KCl, 5.56 mmol/l glucose, 3% BSA, 1 mmol/l MgCl₂, 2 mmol/l CaCl₂, 1 mmol/l MnCl₂) at 37°C for 2 h, and air dried for future use.

A total of 100 µl of HCCLM6 cell suspension (2×10⁵ cells/ml) was plated in each well of an FN-coated 96-well high binding microplate. A total of 100 ml of DMEM medium containing β3 at concentrations of 20, 40, 100 or 200 µmol/l was added at the same time. The final concentrations of β3 were 10, 20, 50 or 100 µmol/l, respectively. The same volume of cell culture medium without β3 was added to the control group and 200 ml of cell culture medium only was added in the plate for the blank group. The assay was conducted in quintuplicate for each sample. Following incubation for 3 h at 37°C in 5% CO₂, the unattached cells were gently washed away with HANKS buffer (Sangon Biotech Co., Ltd.). The attached cell number in each well was measured using the MTT assay (see below). The inhibition rate of β3 on cell adhesion to FN was calculated with the following equation: Cell adhesion inhibition rate = (average OD of control well - average OD of β3-treated well) / (average OD of control well - average OD of blank well) × 100%.

The number of attached cells in each well was analyzed by the MTT assay, and quantified by a micro-titer plate reader (Amersham, USA). Briefly, 100 µl DMEM and 20 µl MTT (5 mg/ml; Sigma-Aldrich) were added to each well. After incubation at 37°C for 4 h, the medium and MTT was discarded. A total of 200 µl of 0.04 mol/l hydrogen chloride-2-propanol solution was added to each well. The amount of MTT formazan product, which reflects the number of cells adhering to FN, was determined by measuring absorbance with a microplate reader at a wavelength of 570 nm and a reference wavelength of 630 nm.

The invasion assay was performed as described previously (19). Briefly, the upper portions of Transwell chambers (Corning, New York, NY, USA) were coated with 75 µl of Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) diluted 1:10 in serum-free DMEM and incubated at 37°C for 2 h. The supernatants of HCCLM6 cells cultured with DMEM containing 10% FCS were harvested once the cells had grown to confluence, and after the addition of FN at a final concentration of 5 µg/ml, resulting in conditioned medium. The cells were harvested by trypsinization and diluted to a 2×10⁶/ml cell suspension with serum-free DMEM. A total of 100 µl of the cell suspension and 100 µl of 200 µmol/l β3 peptide in serum-free DMEM (or serum-free DMEM only as a control) were added in the upper chambers. Then, 600 µl of conditioned medium was added to the bottom chamber of the Transwell plate. After incubation at 37°C for 48 h under a 5% CO₂ atmosphere, the non-invading cells and the gel were gently removed from the upper chamber with cotton-tipped swabs. After rinsing with PBS, cells on the filters were fixed with formaldehyde and stained in Giemsa staining solution for 30 min. The number of invaded cells on the filters were counted in five randomly selected high-powered (×200) fields per filter under a microscope (Leica, Heerbrugg, Switzerland). The invasion inhibition rate was calculated using the following equation: Invasion inhibition rate = [1 - (invaded cell number in β3 chamber / invaded cell number in control chamber)] × 100%.

A total of 24 6-week-old male BALB/cA nude mice were obtained from the Shanghai Institute of Materia Medica, Chinese Academy of Sciences (Shanghai, China). A tumor block from tumor-bearing mice was implanted into the left lobe of the nude mouse liver as described previously (20). Briefly, a left upper abdominal transverse incision was made under anesthesia. The left lobe of the liver was exposed and a part of the liver surface was mechanically injured with scissors. Next, a tumor block of 0.2×0.2×0.2 cm was fixed within the liver tissue. After the surgery, mice were kept in laminar-flow cabinets under pathogen-free conditions and given free access to food and water. Liver cancer early resection was performed at 0.2 cm from the edge of the tumor at day 10 following implantation, prior to metastasis.

Animals were randomly assigned to 2 groups (each group, n=12). At day 1 following resection, the animals were subcutaneously administrated 100 µl of 1 mg/ml of β3 or normal saline (NS) as a control every other day for 10 doses. Half of the mice (n=6) in each group were sacrificed at day 55 post-implantation with an overdose of 6% chloral hydrate (0.5 ml/100 g of body weight; Sigma-Aldrich). At autopsy, the viscera of the animals were examined macroscopically. If recurrence of tumors at the incisal margin was observed, the lesions were resected and weighed.

The other 6 mice of each group were housed in pathogen-free cages. Each animal was examined daily until death. The number of days that each animal survived following resection was recorded. Survival curves were constructed using the Kaplan-Meier method.

All animal experiments were conducted in strict accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. This study was approved by the Experimental Animal Ethics Committee of Shanghai Medical College, Fudan University (approval no. 20021101).

All data were entered into Excel spreadsheets (Excel, Microsoft, Seattle, USA). Statistical analysis was performed using Student's t-test, or the Mann Whitney U test when the data were not normally distributed. Values of P<0.05 in a two-tailed fashion were considered to indicate a statistically significant difference. All analyses were performed using SAS (SAS Institute Inc., Cary, NC, USA).

HCCLM6 cells incubated with 10, 20, 50 and 100 µmol/l β3 for 3 h exhibited a marked reduction in cell adhesion. The adhesion inhibition rates were 11.8, 21.7, 37.5 and 66.4%, respectively (Fig. 1). These findings indicate that β3 is able to inhibit the adhesion of HCCLM6 cells to FN, and β3 may obstruct the invasion of HCC cells to paratumor liver parenchyma.

Following incubation with 100 µmol/l β3, the number of invaded HCCLM6 cells was reduced significantly, with an inhibition rate of 51.3% (Table I). Therefore, β3 may block HCC cells from invading the surrounding tissue and entering and extravasating from the circulation in vivo.

On the 10th day post-tumor-implantation, LCI-D20 tumors were resected, and β3 or the same volume of NS was subcutaneously injected. On day 55, mice were sacrificed to check for intrahepatic recurrence. The recurrent tumors were located around the incisal margins. There were 2 (2/6) mice with intrahepatic recurrent tumors in the β3 group, while there were 6 (6/6) mice with intrahepatic recurrent tumors in the control group (Fig. 2 and Table II). Compared with the control group, the weight of recurrent tumors of the β3 group were markedly reduced. These results indicate that β3 may have inhibitory effects on tumor recurrence at the incisal margin.

Twelve animals were randomly assigned to 2 groups (each group, n=6). At day 1 following resection, the animals were subcutaneously administrated 100 µl of 1 mg/ml of β3 or NS as a control every other day for 10 doses. The mice were housed in pathogen-free cages, and given free access to autoclaved food and water. The survival curves were constructed using the Kaplan-Meier method (Fig. 3). The median survival times after early resection were 136.5 days (control group) and 166.5 days (β3 group), respectively. This result indicates that β3 treatment prolonged the survival time of mice following early resection.