All human esophageal squamous carcinoma cell lines KYSE 170 parental (wild) and derivate cells (KYSE 170mock, KYSE 170F3 and KYSE 170F4) were established in our laboratory and maintained in RPMI 1640 (Life Technologies, Gaithersburg, MD) and Ham's F12 (Nissui Pharmaceutical, Tokyo, Japan) with 2% fetal bovine serum (FBS).
Western blot analysis
Whole-cell extract lysate was prepared from 1 × 107 cells in a sample buffer (2% sodium dodecyl sulfate [SDS], 10% glycerol, 50 mM Tris-Hcl, pH 6.8) at room temperature. Cell lysates were sonicated and the protein content was measured with the Bradford method using BCA Protein Assay Reagent (Pierce, Rockford, MA), after which the cell lysates were electrophoresed on a 12% polyacrylamide gel SDS page and transferred to a polyvinylidene difluoride membrane (Immobilon, Milipore, Bedford, MA) using a semidry transfer blot system (Bio-Rad, Hercules, CA). The membranes were blocked with TBS (20 mM Tris, 150 mM NaCl, pH 7.6) containing 1% Tween 20 and 5% skimmed milk for 1 hour. The membranes were incubated overnight at 4°C with anti-human fascin mouse monoclonal antibody (DAKO, Osaka, Japan; diluted 1:500) or with anti-human β-actin mouse monoclonal antibody (Sigma Inc., St. Louis, MO; diluted 1:2000) as an internal control. They were washed and then incubated at room temperature for 1 hour with anti-mouse IgG-HRP (Zymed, San Francisco, CA), as a secondary antibody, and analyzed using ECL plus reagent (Amersham, Buckinghamshire, UK).
Purification of total cellular mRNA and Reverse Transcription-PCR
Total RNA was extracted from KYSE cell lines by the TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocols [28, 29]. Reverse transcription of total cellular RNA was performed using a First-Strand cDNA Synthesis Kit (Amersham, Buckinghamshire, UK). cDNA was subjected to PCR for 25 cycles of amplification using an Advantage cDNA PCR kit (Becton Dickinson Biosciences, Palo Alto, CA). Each PCR cycle consisted of a denaturation step for 1 minute at 94°C and an annealing step for 1 minute at 60°C. The final extension step was carried out for 5 minutes at 72°C. The PCR products were separated on 1.5% agarose gel and visualized by ethidium bromide staining. PCR primers used for fascin were 5'- AGGCGGCCAACGAGAGGAAC-3' as the forward primer and 5'-ACGATGATGGGGCGGTTGAT-3' as the reverse primer; and for glyceraldehydes-3-phosphate dehydrogenase (G3PDH), 5'-TGGTATCGTGGAAGGACTCATGAC-3' was used as the forward primer and 5'-ATGCCAGTGAGCTTCCCGTTCAGC-3' as the corresponding reverse primer. cDNA from HeLa cells was used as a positive control for each analysis.
Cell growth assay
Cells were plated into 6 cm dishes (2 × 104 cells per dish) at day 0 and incubated for 24 hours for sufficient cell growth. Cells were harvested with trypsin/EDTA every 48 hours for five days, and counted with a cell counter (Coulter Z1, Beckman Coulter, Fullerton, CA). To examine the effect of the suppression of fascin expression on cell growth, we compared it with the control culture in triplicate. Each experiment was repeated three times independently.
Cells were seeded into collagen-coated plates (Becton Dickinson, MA) without FBS. After 24 hours of incubation, the adherent and floating cells were counted from five randomly selected fields. The assay was repeated three times under the same conditions.
200 μmol/L Z-VAD-FMK (BD Biosciences, San Jose, CA) was added to the cells after the cells were plated. The inhibition of cell growth was measured by MTT assay and cell counting assay. The assay was repeated three times under the same conditions.
Construction of fascin-small interfering RNA vector and transfection
In order to construct a vector for fascin-small interfering RNA (siRNA), pSilencer2.1-U6 hygro (Ambion, Inc., Austin, TX) was digested with BglII and HindIII. A chemically synthesized oligonucleotide encoding a fascin-short hairpin siRNA that included a loop motif was inserted downstream of the U6 promoter of the plasmid using a DNA ligation kit (Takara Bio, Inc., Shiga, Japan), and was cloned. Sequences of the oligonucleotide targeted to fascin were 5'-GCCUGAAGAAGAAGCAGAU-3' corresponding to positions 116 to 134 within fascin exon 1. An ESCC cell line KYSE170 was stably transfected with either the fascin-siRNA expression vector or the negative control vector (pSilencer2.1-U6 hygro) using FuGene6 reagent (Roche Diagnostics, Basel, Switzerland) , and cell clones were selected against 100 μg/mL hygromycin (Nacalai Tesque, Kyoto, Japan).
Tumor formation assay in nude mice
Suspensions of 1 × 106 KYSE 170 parental (wild) and derivate cells (KYSE 170mock, KYSE 170F3 and KYSE 170F4) in PBS (60 μL) were injected subcutaneously into the right flanks of 5-week-old male BALB/c nu/nu mice (Japan SLC, Shizuoka, Japan) at day 0. The inoculation was conducted in five mice per group, and tumor growth was estimated from the average volume of tumors by the formula: 1/2 × L2 × W (where L is the length of the tumor and W is the width of the tumor). At 30 days after injection, all mice were sacrificed and the subcutaneous tumors were resected and fixed in 10% formaldehyde/PBS. Tumors were then embedded in paraffin and stained with H&E and fascin. All animal experiments were performed in accordance with institutional guidelines.
Resected tumors from the in vivo experiments were fixed in 10% formaldehyde solution, embedded in paraffin, cut in 4 μm thick sections, and mounted on aminopropyltriethoxylane-coated glass slides. Immunohistochemical staining was carried out using an Envision Kit (Dako Cytomation, Glostrup, Denmark). Tissue sections were incubated overnight at 4°C with anti-human fascin monoclonal antibody clone 55kDa (DAKO, Osaka, Japan; 1:50 dilution) and then incubated with biotinylated anti-mouse IgG for 30 minutes at room temperature. Tissue sections were then stained with 3,3' diaminobenzidine liquid system (Dako Cymation, Glostrup, Denmark), counterstained with Mayer's haematoxilyn, dehydrated and mounted. We performed the same protocol using goat anti-human caspase 3 polyclonal antibody (Santa Cruz Biotechnology, CA, USA; dilution 1:500). As a negative control, the primary antibody was replaced with normal mouse IgG, and a further control was carried out without the primary antibody.
The DeadEnd Colorimetric TUNEL system kit was used for the TUNEL assay. The paraffin-embedded tissues were fixed in 4% formaldehyde with PBS for 15 minutes, permeabilized with 20 μg/ml proteinase K for 15 minutes and subsequently incubated at 37°C for 60 minutes with the rTdT reaction mix on the slide. Streptavidin HRP solution 1:500 in PBS was added and incubated for 30 minutes. DAB solution was then added for 5 minutes. Then the apoptotic cells were counted in five different fields at 40 ×, obtaining the average and standard deviation (SD).
The statistical analysis was performed using the software StatView 4.5 (Abacus Concept, Berkley, CA). A p-value of < 0.05 indicated statistical significance.