Tissue samples and cell lines
Paired (HCC and adjacent non-tumor liver) tissues were obtained from seven patients who underwent liver resection for HCC at Stanford Hospital, Stanford University, California. This study was approved by the Institutional Review Board for the use of human subjects in medical research, and informed consent was obtained from patients prior to liver resection. Ages ranged from 44 to 73 years. All patients were men with HBV-related HCC.
Human hepatoma cell lines, HepG2, Hep40, and Huh7 were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 μg/mL penicillin and 100 μg/mL streptomycin. All media and supplements were from Invitrogen (Carlsbad, CA). Cells were maintained at 37°C in a humidified atmosphere with 5% CO2.
Anti-Wnt-1 antibody and blocking peptide
The anti-Wnt-1 goat polyclonal antibody, the corresponding control goat IgG, and the Wnt-1 specific blocking peptide were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies were concentrated using Microcon-30 ultrafugation devices (Millipore Corporation, Bedford, MA) before being added to cells. The blocking peptide was purified by dialysis using a Slide-A-Lyzer Dialysis Cassette with a 2,000 MWCO (Promega, Madison, WI). For blocking experiments, blocking peptide was pre-incubated with cells overnight, at 20-fold concentration over the anti-Wnt-1 antibody.
Primary culture of hepatocytes
Cryopreserved human hepatocytes, collagen hand-coated 96-well plates, CHRM Thawing Medium, Cell Plating Medium, and Cell Maintenance Medium were received from CellzDirect/Invitrogen (Durham, NC). Characteristics of the three hepatocyte lots are shown in Table . Cryopreserved human hepatocytes were thawed based upon CellzDirect's standard method: hepatocytes were thawed at 37°C, then poured into pre-warmed 37°C CHRM Thawing Medium at a ratio of one vial (approximately 5 million cells)/50 ml in a conical tube. The cells were then centrifuged at 100g for 10 min and resuspended to 0.75 × 106 cells/mL in Plating Medium. Cell viability was determined by trypan blue exclusion. Hepatocytes were then plated in collagen-coated 96-well plates at a density of 3 × 104 cells/well in a volume of 100 μl/well. After 5 h of incubation at 37°C, Plating Media was replaced with serum-free Maintenance Media at 100 μl/well, and incubated overnight for cytotoxicity assay as described below.
Characteristics of tested normal hepatocytes
Cell viability and proliferation assays
Hepatoma cells were seeded in 96-well plates at 3 × 103 cells/well, and incubated overnight at 37°C prior to addition of anti-Wnt-1 antibody. Anti-Wnt-1 antibody and control IgG were added at desired final concentrations (range from 0-20 μg/ml), and further incubated for 72 h before cell viability and proliferation were assessed using CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI) or .CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI) respectively, according to the manufacturer's instructions. Briefly, for the .CellTiter-Glo assay, the assay regents (a combination of detergent and luciferase-based enzyme) were directly added to cultured cells, resulting in cell lysis and the production of a bioluminescent signal proportional to the amount of ATP present. Luciferase activity was measured on a luminometer (Berthold LB-96V) and values were normalized to the ATP activity and compared with their respective PBS control value, which was set at 100. Three independent experiments were done, each in triplicates. For proliferation assay, optical density (OD) was read at 490 nm using a SAFIRE microplate reader (TECAN, Research Triangle Park). The background value (mean OD values from wells with only the AQueous One Solution) was subtracted from all data. Three independent experiments were done, each in triplicates.
Luciferase reporter gene assay
β-catenin/Tcf4 transcriptional reporter gene assays were performed using TCF/Luc reporter constructs, wild type pTOPFLASH, and mutant pFOPFLASH, which were generously provided by B Vogelstein (John Hopkins Oncology Center, Baltimore, MD, USA) [34
]. Cells were seeded at 3 × 104
cells/well into 24-well plates and incubated for 24 hr prior to transfection with wild type pTOPFLASH or mutant pFOPFLASH (0.7 μg) using Lipofectamine 2000 (Invitrogen, Carslbad, CA) according to the manufacturer's instructions. The β-galactosidase (β-gal) expression vector (0.1 μg) was added to each transfection system to normalize the transfection efficiency. After 4 hr, medium containing transfection regent was replaced with new culture medium containing the anti-Wnt-1 antibody or control IgG (2 μg/ml). After 48 hr, cells were lysed in 100 μl of lysis buffer, and 20 μl aliquots were assayed for luciferase activity using the Promega Luciferase assay system or for β-gal activity using the Promega β-gal assay system. Relative light units (RLU) were measured and normalized for transfection efficiency using β-gal activity. Final RLU representing Tcf4 transcriptional activity were calculated by subtracting normalized levels obtained with pFOPFLASH from those obtained with pTOPFLASH.
TUNEL (Terminal dUTP nick-end labeling) assays (Promega, Madison, WI) were performed according to the manufacturer's protocol. Briefly, Huh7 or Hep40 cells were seeded in 8-chamber BD tissue culture slides (BD Bioscience Labware, Bedmord, MA) at 10% confluency. Control IgG or anti-Wnt-1 antibody was added to the medium at final concentration of 10 μg/ml each. After 72 hr incubation, cells were washed twice with PBS, and then fixed in 4% paraformaldehyde for 25 min. Fixed cells were washed twice in PBS with 0.1% Triton X-100, and then incubated with TUNEL reaction mixture for 60 min at 37°C. After washing with 2xSSC, slides were immersed in PBS with 1 μg/ml Propidium Iodide (PI) for 5 min in the dark and then washed with PBS. Fluorescence labeling was visualized and photographed (100× magnification) with a fluorescence microscope (Nikon Eclipse 80i, Nikon Corporation, Tokyo, Japan) and with a digital camera (Nikon DXM1200f, Nikon Corporation, Tokyo, Japan). For TUNEL staining of the tumor xenografts, 4-μm tissue sections of tumor xenografts from in vivo experiments were stained using the ApopTag Peroxidase in Situ Oligo Ligation Apoptosis Detection Kit (Chemicon International, Temecula, CA) according to the manufacturer's protocol.
Western blotting and antibodies
Huh7 or Hep40 cells were seeded at 50% confluency in 6-well plates and incubated at 37°C overnight. Cells were then treated with PBS, control IgG, or anti-Wnt-1 antibody (2 μg/ml) for 48 hr. Cell monolayers were washed twice with PBS and then lysed in RIPA extraction buffer. For nuclear β-catenin immunodetection, nuclear extracts were prepared with a NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce, Rockford, IL). Equal amounts of protein (20 μg) were resolved by SDS-PAGE and Western blots were performed by using the primary antibodies to c-Myc (1:500, Cat. 551101, BD Pharmingen, San Diego, CA), cyclin D1 (1:1000, Cat. ab6152, Abcam, Cambridge, MA),, survivin (1:1000, Cat. NB500-201H, Novas Biologicals, Littleton, CO), β-catenin (1:500; Cat. SC-7963, Santa Cruz Biotechnology, Santa Cruz, CA), Histone H3 (1:10000, Cat. Ab21054, Abcam, Cambridge, MA) or β-actin (1:10000, Cat.A3854, Sigma-Aldrich, MO). Secondary antibodies (anti-mouse, Cat.SC-2005, and anti-rabbit, Cat.SC-2004) conjugated with horseradish peroxidase were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Western blots were analyzed by ImageJ software, and signal intensities normalized to β-actin.
Xenografts in Nude Mice
Nude mice (ATHYMIC NU/NU; Harlan Sprague-Dawley, Indianapolis, IN) at age 4-6 weeks with a body weight of 18 to 25 g were used for the experiments. Mice were injected subcutaneously at the dorsal region with 5 × 106/150 μl viable Huh7 cells. After two weeks, when tumors reached approximately 0.4-0.5 cm in diameter, mice were randomized into groups (n = 5) to be intratumorally injected with 100 μl of PBS or anti-Wnt-1 antibody at the dose of 50 μg/kg once a week. Tumor size was measured with digital calipers every three days and was calculated using the formula π/6 × larger diameter × [smaller diameter]2. Mice were sacrificed at the end of the treatment period, and xenografts harvested.
Immunoperoxidase stain of tumor xenografts was performed on acetone-fixed 4-μm tissue sections. Briefly, sections were incubated with monoclonal mouse anti-human c-Myc (1:200, Cat. 551101, BD Pharmingen, San Diego, CA), cyclin D1 (1:250, Cat. ab6152, Abcam, Cambridge, MA), or survivin (1:500, Cat. NB500-201H, Novas Biologicals, Littleton, CO) and then washed with PBS. Subsequent procedures were performed using Dakocytomation Envision System-HRP mouse system (DakoCytomation Inc, CA, USA) according to the manufacturer's protocol. We observed c-Myc staining in the cytoplasm and nuclei, and cyclin D1 and survivin staining in the cyptoplasm, consistent with other reports in HCC [35
]. Therefore, for quantification, 100 cells at 3 randomly selected areas were assessed, and the number of cells that stained positively for c-Myc, cyclin D1, or survivin were counted.
Statistical analysis was performed by one-way ANOVA and independent-sample T-test using the computer SPSS software. In all assays, the probability value (P) of < 0.05 was considered statistically significant.