Cell lines and primary cultures of hepatocytes
Human hepatoma cell lines (Huh7, HepG2, Hep40) 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.
Cryopreserved human hepatocytes, collagen-coated 96-well plates, CHRM Thawing Medium, Cell Plating Medium, and Cell Maintenance Medium were received from CellzDirect/Invitrogen (Durham, NC). Characteristics of the hepatocytes and the plating method are as described previously .
Human Wnt3 cDNA clone pUSEamp-Wnt3 (HA-Wnt3) and the empty vector pUSEamp were from Millipore (Billerica, MA). pCMV6-XL5-FZD7 and the empty vector pCMV-XL5 were from OriGene Technologies (Rockville, MD). TCF/Luc reporter constructs, pTOPFLASH (wild type) and pFOPFLASH (mutant), were from B Vogelstein (John Hopkins Oncology Center, Baltimore, MD, USA). The pet28A-sFZD7 was constructed by cloning the PCR-amplified product of the sFZD7 coding region into the BamHI and HindIII sites of vector pet28A (Merck Biosciences, Darmstadt, Germany). sFZD7 fragment was amplified using template containing full length FZD7 plasmid with the following primers: upstream (5'-TTTGGATCCTGCCAGCCCATCTCCAT-3'), and downstream (5'-TTTAAGCTTCACCGGGTGCGGGCGAAGCGCCTCT-3'). The PCR reaction was performed in a GeneAmp PCR system 2700 (Applied Biosystems, Foster City, CA) under the following conditions: heat activation of the polymerase for 5 min at 94°C, followed by 30 cycles of 95°C for 30 sec, 58°C for 30 sec, and 72°C for 45 sec; with a final extension at 72°C for 10 min.
Expression and purification of sFZD7
Recombinant His-sFZD7 peptide was produced in E. coli (strain BL21, DE3) with IPTG induction, and purified from the insoluble fraction of the bacterial lysate with Ni-NTA (Qiagen, Carpinteria, CA) following the manufacturer's instructions. Elution was done with 8 M urea elution buffer (pH 4.0). Eluted His-sFZD7 fusion protein was dialyzed against phosphate-buffered saline (PBS) with a stepwise increasing pH gradient (pH 4.0 to pH 7.4) at 4°C. The concentration of final His-sFZD7 fusion peptide was determined by BCA protein assay (PIERCE, Rockford, IL) and purification was confirmed by SDS-PAGE and immunoblotting using HRP-labeled anti-His tag antibody (1:1000, Abcam, Cambridge, MA).
Pull down assay
Huh7 cells (5 × 106) were lysed in RIPA buffer and the crude lysate was clarified by centrifugation at 12,000 × g for 5 min. Recombinant His-sFZD7 peptide (100 μg) was mixed with 15 μl Ni-NTA agarose beads (Qiagen, Carpinteria, CA) in binding buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) and incubated for 1 h with gentle rotation, and then washed with wash buffer (50 mM NaH2PO4, 300 mM NaCl, 40 mM imidazole, pH 8.0). The Ni-NTA agarose with recombinant His-sFZD7 peptide was mixed with Huh7 cell lysate; mock agarose was separately mixed with Huh7 cell lysate. Imidazole was added to the mixtures at a final concentration of 40 mM, and incubated overnight with gentle rotation in 4°C. After centrifugation at 400 × g for 2 min at 4°C, supernatants were discarded and the agarose mixtures were washed with binding buffer three times for 5 min each. The agarose mixtures with the protein complex were then eluted with elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0) and the supernatants were resolved on SDS-PAGE. Western blots were done using the rabbit anti-Wnt3 (1:1000, Abcam, Cambridge, MA), and anti-His tag (1:1000, Abcam, Cambridge, MA) antibodies.
Luciferase reporter gene assay
Huh7 cells (at 50% confluency) were transfected with 0.2 μg of HA-Wnt3 or empty vector and 0.2 μg of pCMV6-XL5-FZD7 or the empty vector, along with 0.3 μg of pTOPFLASH or pFOPFLASH reporter plasmids and 0.1 μg of β-gal to normalize for transfection efficiency. The amount of DNA in each transfection reaction was kept constant by adding an appropriate amount of the empty vector. Transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. After 4 h, media containing transfection reagent were replaced with new culture media with or without sFZD7 (2 μg/ml). After 48 h, cells were lysed in 100 μl of lysis buffer, and 20 μl aliquots were assayed using the Promega Luciferase assay system, and 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.
Protein extracts and immunoblotting
Huh7 and HepG2 cells were seeded at 50% confluency in 6-well plates and incubated at 37°C overnight. Cells were then treated with PBS, or with purified sFZD7 at desired concentrations (2, 10, or 20 μg/ml) for 48 h. Cell monolayers were washed twice with PBS and then lysed in the 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. No. 551101, BD Pharmingen, San Diego, CA), cyclin D1 (1:1000, Cat. No. ab6152, Abcam, Cambridge, MA), survivin (1:1000, Cat. No. NB500-201H, Novas Biologicals, Littleton, CO), β-catenin (1:500; Cat. No. SC-7963, Santa Cruz Biotechnology Inc., Santa Cruz, CA), HRP-Histone H3 (1:10000, Cat. No. Ab21054, Abcam, Cambridge, MA) and HRP-β-actin (1:10000, Cat. No. A3854, Sigma-Aldrich, MO). Secondary antibodies (anti-mouse, Cat. No. SC-2005 and anti-rabbit, Cat. No. SC-2004) conjugated with horseradish peroxidase were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
For sFZD7 and doxorubicin combination treatment, Huh7 and HepG2 cells were treated with PBS, purified sFZD7 alone (10 μg/ml for 48 h), doxorubicin alone (2.5 μM for 6 h), or combination of sFZD7 and doxorubicin (10 μg/ml of sFZD7 for 48 h, followed by addition of 2.5 μM doxorubicin for 6 h). Cell monolayers were then lysed in the RIPA extraction buffer. Equal amounts of total cell protein (20 μg) were resolved by SDS-PAGE and Western blots were performed by using the primary antibodies to ERK1/2 (1:1000, Cat. No. 9102, Cell Signaling Technology Inc., Danvers, MA), p-ERK1/2(1:1000, Cat. No. 4370, Cell Signaling Technology Inc., Danvers, MA), AKT (1:1000, Cat. No. 9272, Cell Signaling Technology Inc., Danvers, MA), p-AKT (1:1000, Cat. No. 4058s, Cell Signaling Technology Inc., Danvers, MA).
Quantitative real-time PCR assay
Total RNA was extracted from hepatoma cells and hepatocytes by using the RNeasy mini kit (Qiagen, Valencia, CA) following the manufacturer's instructions. Concentration and purity of extracted RNA were determined by optical density measurement at 260 and 280 nm. All real-time PCR reagents were from Applied Biosystems (Foster city, CA). Briefly, first-strand cDNA was generated by random primers using Taqman Reverse Transcriptional Reagent, and real-time PCR was performed by using Taqman Gene Expression Assay (Human FZD7 assay ID: Hs00275833_s1; Human Wnt3 assay ID: Hs00229135_m1) and Universal PCR Master Reagent in a Stratagene Mx3000P Q-PCR system (Stratagene, La Jolla, CA). These reactions were incubated at 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec, and 60°C for 1 min. The expression level of FZD7 was measured in terms of threshold cycle value using Stratagene MxPro software and normalized to the internal control, human 18s rRNA (Part No. 4333760F).
Cell viability assay
Hepatoma cells were seeded in 96-well plates at 3 × 103 cells/well, and normal hepatocytes seeded at 3 × 104 cells/well, and incubated overnight at 37°C prior to addition of sFZD7 at desired final concentrations (range from 1.25-20 μg/ml). Cells were further incubated for 72 h before cell viability was assessed using CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI) according to the manufacturer's instructions. Luciferase activity was measured on a luminometer (Berthold LB-96V) and values were normalized to the ATP activity and compared with the PBS control value, which was set at 100. Three independent experiments were done, each in triplicates.
Cell proliferation assay
Hepatoma cells were seeded in 96-well plates at 3 × 103 cells/well, maintained overnight at 37°C, and incubated with doxorubicin (at various concentrations, ranging from 0-100 μM) with or without sFZD7 (2 μg/ml). After 72 h incubation, cell proliferation was monitored by using CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI) according to the manufacturer's instructions. Optical density (OD) at 490 nm was read using a microplate reader (BioTek Instruments, Inc., Winooski, VT). The 50% inhibitory concentrations (IC50s) were calculated as an estimate of the anti-proliferative effects of doxorubicin alone or in combination with sFZD7.
Xenografts in nude mice
Animal experiments were approved by the Administrative Panel on Laboratory Animal Care at Stanford University. Nude mice (ATHYMIC NU/NU; Harlan Sprague-Dawley, Indianapolis, IN) at age 4-6 weeks (body weight of 18 to 25 g) were used. Mice were injected subcutaneously at the dorsal region with 5 × 106 viable Huh7 cells. After two weeks, when tumors reached approximately 0.4-0.5 cm in diameter, mice were randomized into four groups (n = 5 each) to be intratumorally injected with (1). PBS control (100 μl, once weekly); (2). purified sFZD7 (dose:12.5 mg/kg; volume: 100 μl; once weekly); (3). Doxil (dose: 2.5 mg/kg; volume: 100 μl; once weekly); or (4). sFZD7 (dose: 12.5 mg/kg; volume: 100 μl; once weekly) plus Doxil (dose: 2.5 mg/kg; volume: 100 μl; once weekly). Doxil is a liposomal formulation of doxorubicin for intravenous use. Intratumor administration was chosen to mimic the clinically used treatment method of chemoembolization, which delivers cytotoxic drugs directly to the HCC tumor for greater efficacy and reduced toxicity. Tumor size was measured with digital calipers every three days and was calculated using the formula π/6 × larger diameter × [smaller diameter]2. Mice in the PBS group were sacrificed after 14 days, whereas mice in other treatment groups were sacrificed after 17 days of treatment. Tumor tissues were harvested for immunohistochemistry as described below, and for TUNEL staining using the ApopTag Peroxidase in Situ Oligo Ligation Apoptosis Detection Kit (Chemicon International, Temecula, CA) according to the manufacturer's protocol. We assessed the percentage of cells that stained positively with brown nuclei in five randomly selected areas of 100 cells per area. Western blot was used to quantify the protein levels of c-Myc, cyclin D1, or survivin in tumor xenografts of two mice in each group. The antibodies used were: HRP-conjugated anti c-Myc (1:1000, Cat. No. ab62928, Abcam, Cambridge, MA), biotin-conjugated anti-cyclin D1 (1:1000, Cat. No. MS-210-B0, Thermo Scientific, Fremont, CA), HRP-conjugated anti-survivin (1:1000, Cat. No. NB500-201H, Novas Biologicals, Littleton, CO), HRP-conjugated β-actin (1:10000, Cat. No. A3854, Sigma-Aldrich, St. Louis, MO), and secondary antibodies (anti-biotin) conjugated with HRP (1:1000, Cat. No. Ab19221-1, Abcam, Cambridge, MA). Western blots were analyzed by ImageJ software, and signal intensities normalized to β-actin.
Immunoperoxidase staining of tumor xenografts was performed on formalin-fixed 4-μm tissue section. Briefly, sections were incubated with monoclonal mouse anti-human c-Myc (1:200, Cat. No. 551101, BD Pharmingen, San Diego, CA), cyclin D1 (1:250, Cat. No. ab6152, Abcam, Cambridge, MA), or survivin (1:500, Cat. No. NB500-201H, Novas Biologicals, Littleton, CO) and then washed with PBS. Subsequent procedures were performed using Dakocytomation Envision System-HRP mouse system (Dako, Carpinteria, CA) according to the manufacturer's protocol.
Statistical significance was determined by the independent-sample T-test using the computer SPSS software (version 10.0; SPSS, Chicago, IL). P values less than 0.05 were considered statistically significant.