KITENIN Is Associated with Tumor Progression in Human Gastric Cancer

Materials and Methods

Patients and tumor specimens. Twenty fresh gastric cancer tissues and paired normal tissues were collected and immediately frozen in liquid nitrogen for RNA and protein preparation by gastrointestinal endoscopic biopsy at Chonnam National University Hwasun Hospital (Jeonnam, Korea) between May 2009 and June 2009. For evaluating the impact of KITENIN on patient prognosis including survival, paraffin tissue sections and clinical data were collected from 80 consecutive patients who underwent surgery for gastric cancer at Chonnam National University Hwasun Hospital between April 1997 and December 1997. None of the patients had received preoperative radiotherapy or chemotherapy. All had primary tumor resection with regional lymph node dissection. Formalin-fixed and paraffin-embedded tissue blocks were selected by viewing the original pathologic slides and choosing blocks that showed the junction between normal gastric epithelium and tumor. Tumor staging was in accordance with the American Joint Committee on Cancer (AJCC) staging system (20). Survival was measured from the time of surgery until follow-up at December 2008. This study group comprised 58 males and 22 females. The mean age was 56.9±10.6 (mean±SD), with a range from 33 to 82 years. The mean size of tumor was 4.9±3.3 cm (mean±SD), with a range from 0.5 to 20.0 cm. The mean follow-up period was 78.7 months, with a range from 0.2 to 140.8 months. This study was approved by the Ethics Committee of Chonnam National University Hwasun Hospital in Jeonnam, Korea.

Cell culture and transfection. AGS cells were obtained from the Korean Cell Line Bank (Seoul, South Korea) and grown in RPMI-1640/25mM HEPES (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone), 50 units/ml penicillin, and 50μg/ml streptomycin (Gibco, Grand Island, NY, USA). Cells were incubated in a water-saturated atmosphere of 5% CO₂ at 37°C. For experiments, cells were seeded on plates such that they would be 40~50% confluent at the time of transfection. The synthesized human KITENIN siRNA (5′-GCUUGGACUUCAGCCUCGUA GUCAA-3′) and negative control siRNA (Qiagen, MD, USA) were transfected with Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA). The KITENIN knockdown was identified by RT-PCR and Western blotting.

Cell invasion assay. Transwell filters (8.0 μm pores) were coated with 1% gelatin/RPMI-1640 overnight and dried out at room temperature. Cells transfected with siRNA were seeded at 2×10⁵ cells in 120 μl 0.2% bovine serum albumin (BSA) medium in the upper chamber. Subsequently, 400 μl 0.2% BSA medium containing 20 μg/ml human plasma fibronectin (Calbiochem, La Jolla, CA, USA), a chemotactic factor, were loaded into the lower chamber. After incubation for 24 h, migrated cells on bottom surface of the transwell were stained with Diff Quik solution (Sysmex, Kobe, Japan) and counted in 5 selected fields under a light microscope. Data were expressed as the mean±standard error (SE) of the number of cells/field in three individual experiments.

Cell migration assay. Cells were cultured in a 6-well plate and were transfected with 100 pM of KITENIN and negative control siRNA. After incubation for 48 h, a scratch wound was made by creating a linear cell-free region using a 200 μl pipette tip resulting in a uniform gap. The medium was changed to 5% FBS/RPMI-1640 media. The progress of cell migration into the scratch was photographed at 0, 6, 12, and 24 h using an inverted microscope. The distance between gaps was normalized to 1 cm after capture of six random sites.

Cell proliferation assay. Cells were plated onto a 96-well plate at a density of 10³ cells/well and the next day, cells were transfected with 1 pM of KITENIN and negative control siRNA. After incubation for 48 h, cell proliferation was determined by EZ-CyTox (tetrazolium salts, WST-1) cell viability assay kit (Daeil lab Inc, Seoul, South Korea). After WST-1 reagent was added for 1-2 h at 37°C, the absorbance at 460 nm was determined using a microplate reader (Infinite M200; Tecan, Austria GmbH, Austria) with Magellan V6 data analysis software (Tecan). Triplicate wells were used for each experimental condition and all experiments were repeated at least three times.

RNA isolation and amplification by RT-PCR. RNA from AGS cells and gastric tissues was isolated using the TRIzol method (Invitrogen), reverse transcribed, and amplified as previously described using specific primers for matrix metalloproteinase-1 (MMP-1), MMP-3, cyclin D1, cyclooxygenase-2 (COX-2), c-myc, CD44, KITENIN and GAPDH. Primer sequences were as follows: MMP-1, 5′-TTG GGC TGA AAG TGA CTG G-3′/5′-CCT CCA TTA CCT GGG CCT GG-3′; MMP-3, 5′-ACA GGA TTG TGA ATT ATA CAC-3′/5′-GTG GCC AAT TTC ATG AGC AGC-3′; cyclin D1, 5′-GAC CAT CCC CCT GAC GGC CGA G-3′/5′-CCG CAC GTC GGT GGG TCT GC-3′; COX-2, 5′-GCA TTC TTT GCC CAG CAC TT-3′/5′-AGA CCA GGC ACC AGA CCA AAG-3′; c-myc, 5′-GAG GCC ACA GCA AAC CTC C-3′/5′-GGT CAC GCA GGG CAA AAA AGC T-3′; CD44, 5′-ATG GAC AAG TTT TGG TGC AC-3′/3′-CTT CTA TGA ACC CAT ACC TGC-3′; KITENIN, 5′-AAG CCT TCA TCC ACA TCC AGC-3′/5′-GAG AGC CAT CGA TCC TTG TCA-3′; GAPDH, 5′-ACC ACA GTC CAT GCC ATC AC-3′/5′-TCC ACC ACC CTG TTG CTG TA-3′. For each sample, 1 μg total RNA was used to prepare cDNA in a reaction containing 50 ng/μl oligo-dT (Promega, Madison, WI, USA) and after incubation at 72°C for 10 min, MMLV reverse transcriptase (Invitrogen) and RNAsin (Takara, Otsu, Shiga, Japan) were added and incubation continued at 42°C for 1 h and at 72°C for 15 min. PCR amplification of cDNA was performed using gene-specific primers and GoTaq® DNA polymerase (Promega). PCR products were separated by electrophoresis on 1% agarose gel containing ethidium bromide.

Protein isolation and Western blotting. Total proteins from AGS cells and gastric tissues were prepared using RIPA buffer (1 M Tris-HCl, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA) with 1 mM phenylmethanesulfonyl fluoride (PMSF), Halt™ Phosphatase inhibitor and Halt™ Protease inhibitor cocktail (Thermo, Rockford, IL, USA). Protein concentrations were determined by BCA™ protein assay (Thermo). Total proteins (10-20 μg) were subjected to 10% SDS-polyacrylamide gels and then electrophoretically transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After blocking with 5% BSA in TBS-Tween 20 buffer (TBST) at room temperature for 1 h, PVDF membranes were sequentially blotted with primary antibodies: polyclonal anti-human KITENIN, extracellular signal-regulated kinase1/2 (ERK1/2), phospho-ERK1/2, c-Jun N-terminal kinase (JNK), phospho-JNK, p38, phospho-p38 (Cell Signaling, Danvers, MA, USA) and polyclonal anti-human GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C for overnight. After rinsing in TBST, PVDF were incubated with anti-rabbit or anti-mouse horseradish peroxidase (HRP) secondary antibody (Santa Cruz Biotechnology) at room temperature for 1 h. The blot was detected with chemiluminescence (ECL) HRP substrate (Millipore) and recorded by an image reader (Ras-4000; Fujifilm, Tokyo, Japan).

Luciferase reporter assay. AGS cells were plated in 24-well plates at a density of 5×10⁴ cells. The next day, cells were co-transfected with 50 ng of the reporters (AP-1-luc), 1 ng of phRL-CMV (cytomegalovirus) and 10-20pM of siRNA by Lipofectamine™ 2000 (Invitrogen). After incubation for 48 h, luciferase activity in 20 μl cell extract was measured with a Dual-Luciferse Reporter Assay System (Promega), that of Renilla luciferase activity was assayed to control for transfection efficiency. Two separate experiments were carried out in triplicate. Relative luminescence readings were normalized to Renilla activity and fold induction of reporters by effectors was calculated in comparison with the negative control siRNA.

Immunohistochemistry. Paraffin tissue sections from parents were deparaffinized, rehydrated and retrieved with retrieval buffer. To block the endogenous peroxidase activity, tissues were treated with purchased Peroxidase-Blocking solution. (Dako, Carpinteria, CA, USA) and were incubated with polyclonal rabbit anti-human KITENIN in primary diluent solution (Invitrogen) for overnight at 4°C. After washing in TBST, tissues were stained using Dako Real™ Envision HRP/DAB detection system (Dako). Stained tissues were viewed and photographed using a light microscope.

Evaluation of KITENIN expression. Assessment of the staining for KITENIN was evaluated by two independent observers without knowledge of the clinicopathological data. Consensus scores were assigned for each case by reviewing the slides with discrepancies in scoring. All sections on which the two observers disagreed were re-evaluated and discussed. There was total agreement on the classification. Staining intensity was classified from zero (no staining) to 3 (strong staining), and percentage of staining area was classified as 0 for no positive staining of tumor cells, 1 for positive staining in <10% of the tumor cells, 2 for positive staining in 10% to 50% of the tumor cells, or 3 for positive staining in >50% of the tumor cells. A staining index was calculated as the product of staining intensity and staining area. The tumors were categorized as positive expression (staining index ≥4) or negative expression (staining index <4).

Statistical analysis. Experimental differences were tested with Student's t-test. The χ²-test and Fisher's exact test, where appropriate, were used to compare expression of KITENIN with various clinicopathological parameters. Actuarial survival rates of patients were evaluated according to the Kaplan-Meier method and the differences were tested with a log-rank test. The statistical software program used was the Statistical Package for the Social Sciences (Version 15.0; SPSS, Chicago, IL, USA). A p-value<0.05 was considered statistically significant.