Patients and tissue samples
Patients with pathologically proven hypopharyngeal squamous cell carcinoma (all primary cases) hospitalized at the Department of Otorhinolaryngology at Chiba University Hospital were investigated in this study. Written informed consent was obtained from each patient prior to surgery.
The D562 pharyngeal cancer cell line was purchased from Human Science Research Resources Bank, Osaka, Japan. TE4, TE9, and TE11 human esophageal cancer cell lines were provided by RIKEN BRC (National Bio-Resource of the MEXT, Tsukuba, Japan) [32, 33]. The human esophageal cancer cell lines YES5, TE2, and TTn were kindly provided by Dr. Shimada . All cell lines were cultured at 37°C in a humidified atmosphere containing 5% CO2 and maintained in IMDM (Iscove's modified Dulbecco's medium; Gibco BRL, NY, USA) in tissue flasks supplemented with 10% heat-inactivated FBS and penicillin (100 units/mL) as well as streptomycin (0.1 mg/mL).
Transient siRNA transfection
Double-stranded siRNA oligonucleotides against PPL and firefly luciferase (GL2), the negative control, were purchased from QIAGEN (Hilden, Germany). Cells were grown to 30-40% confluence in 24-well plates, and 20 pmol of siRNA oligonucleotides was transfected using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). Cells were processed for each assay, and whole-cell proteins were extracted 48-72 h after transfection. Knockdown efficiency of PPL by siRNA was assessed by immunoblotting.
Cells were dissolved in lysis buffer (7 M urea, 2 M thiourea, 2% 3-[(3-cholamidopropyl)dimethylammonio-]1-propanesulfate, 0.1 M dithiothreitol, 2% IPG buffer (GE Healthcare, Buckinghampshire, UK), and 40 mM Tris) using a Polytron homogenizer (Kinematica, Switzerland) following centrifugation (100,000 × g) for 1 h at 4°C. The amount of protein in the supernatant was measured by protein assay (Bio-Rad, Hercules, CA, USA). The proteins were separated by electrophoresis on 7.5% polyacrylamide gels and transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA) in a tank transfer apparatus (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 0.5% skim milk in phosphate-buffered saline (PBS) or 0.5% bovine serum albumin (BSA) in tris-buffered saline with Tween 20 (TBST) for 1 h, and probed with a primary antibody diluted in blocking buffer. Anti-PPL (Betyl Laboratory Ltd., Montgomery, TX, USA), anti-phospho-AKT (Ser473 or Thr308), anti-phospho-MAPK (Cell Signaling Technology, Beverly, MA, USA), and anti-β-actin (Santa Cruz, CA, USA) were used as primary antibodies. Donkey anti-rabbit IgG horseradish peroxidase (HRP) conjugate (Amersham Pharmacia Biotech, Piscataway, NJ, USA) diluted 1:3,000 and rabbit anti-goat IgG HRP (Cappel, West Chester, PA, USA) diluted 1:500 were used as secondary antibodies. Antigens on the membrane were detected by ECL™ detection reagents (GE Healthcare, Buckinghamshire, UK).
Five thousand cells were seeded in 24-well Falcon 3072 plates (Becton Dickinson, Lincoln Park, NJ, USA) at 37°C and 5% CO2. After various periods, cell number was established by counting with a phase-contrast Leitz microscope (MD, USA).
For cell cycle analysis, 1 × 106 cells were seeded in 10-cm-diameter culture dishes. Cells were trypsinized, washed with ice-cold PBS, fixed in 70% ethanol, and stored at -20°C. The cells were then treated with RNase (0.2 mg/mL) for 0.5 h at 37°C and stained with propidium iodide at 50 μg/mL. The stained cells were analyzed in a FACSCalibur cytometer (Becton Dickinson, Lincoln Park, NJ, USA), and the results were analyzed with FlowJo software (Tree Star Inc., San Carlos, CA, USA).
BrdU incorporation assay
The BrdU incorporation assay was carried out as described previously, using the BrDu flow kit (Becton Dickinson, Lincoln Park, NJ, USA) according to the manufacturer's instructions . In brief, 1-2 × 106 cells were labeled with 10 μM BrdU for 1 h, harvested, fixed, and treated with DNAase. Cells were stained with anti-BrdU FITC-coupled antibody and 7-AAD for DNA. Samples were analyzed in a FACSCalibur cytometer (Becton Dickinson, Lincoln Park, NJ, USA) using the FlowJo program (Ashland, OR, USA). BrdU signals were recorded on a logarithmic scale in the FL1 channel, and DNA was recorded on a linear scale in the FL3 channel. Single cell events were distinguished from cell aggregates on a FL3-A versus FL3-W plot of 7-AAD fluorescence.
Cells were transfected with PPL siRNA or pretreated for 2 h with PI3K inhibitor, LY294002 (Wako, Osaka, Japan). Then, cells were grown to confluence in 6-well plates, and monolayer cells were scraped using a micropipette (yellow) tip. After washing with PBS, serum-free medium was added to prevent cell proliferation. Photographs of the wounded area were taken immediately after the scratch was made. Six and 24 h after scraping, cell movement into the wounded area was monitored by time-lapse microscopy.
Adhesion of PC-3 cells was measured as described previously, with some modifications [36, 37]. In brief, each well of a 24-well plate was coated with 200 μg/ml Matrigel (Becton Dickinson, Lincoln Park, NJ, USA). In each well, 2 × 104 cells were added in 0.5 mL serum-free media supplemented with 0.1% BSA. The plates were incubated at 37°C and adhesion was determined at 0.5 h. The plates were fixed with methanol and stained with Diff-Quik (Sysmex Corp., Kobe, Japan). The number of cells adhered to the Matrigel was counted at 4 random fields per well in duplicate at a magnification of ×400.
Time-lapse recordings of cells were made with an Axiovert 200 M SP LSM 510 META confocal laser scanning microscope (Carl Zeiss Inc., Oberkechen, Germany) equipped with an AxioCam charge-coupled device (CCD) camera (Zeiss, Germany) and AxioVision software. Cells were maintained in an environmental chamber at 37°C with 5% CO2 during the analysis. Images were taken by CCD camera through a laser scanning microscope every 5 min. Cell tracking was performed using AxioVision software. Images were imported into Adobe Photoshop and prepared as pictures.
Immunohistochemical procedures for PPL expression have been described previously . In brief, air-dried 4-micron cryostat sections were fixed in cold acetone for 20 min and washed with PBS for 5 min. The sections were then incubated with anti-PPL antibody overnight at 4°C. After washing with PBS, the sections were incubated with biotinylated anti-rabbit IgG as a secondary antibody. The sections were then incubated with HRP-conjugated streptavidin-biotin complex for 0.5 h. Peroxidase activity was visualized by DAB solution (20 mg of 3,3'-diaminobenzidine, 65 mg of sodium azide, and 10 mL of 30% hydrogen peroxide in 100 mL of 0.05 M Tris buffer at pH 7.6). Hematoxylin was used for nuclear counterstaining.
The abovementioned experiments were repeated at least 3 times. Student's t-test was used for statistical analysis.