COX-2 Overexpression Induced by Gene Transfer Reduces Sensitivity of TE13 Esophageal Carcinoma Cells to 5-Fluorouracil and Cisplatin

Materials and Methods

Cell culture. The human ESCC cell line, TE13, was obtained from the Cell Resource Center for the Biomedical Research Institute of Development, Aging, and Cancer (Tohoku University, Sendai, Japan). The cells were maintained in culture medium (CM) consisting of RPMI-1640 (Nacalai Tesque Inc., Kyoto, Japan) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Cell Culture Bioscience, Nichirei Biosciences, Tokyo, Japan), and incubated at 37°C in 5% CO₂.

Establishment of COX-2 transfectants. Twenty-four hours after plating TE13 cells (1×10⁵ cells/well) on 24-well plates in CM, cells were transfected with a COX-2 constitutive expression vector (pBOSNeoCOX-2) and control vector (pBOSNeo) (15) using lipofectamine (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. After 24 h, the transfected cells were trypsinized from the plate and placed in a 10-cm culture dish in CM containing a final concentration of 1.0 mg/ml G418 (Geneticin; Invitrogen). The G418-resistant clones were then selected and expanded. The clones, which were confirmed to overexpress COX-2 by the methods described below [western blotting and prostaglandin-E₂ (PGE₂) assay], were used for chemosensitivity testing. The cells transfected with pBOSNeo were used as controls.

Enzyme immunoassay for PGE₂. Cells were cultured overnight in a 12-well plate at a density of 1×10³ cells/well, and incubated at 37°C for 15 min with 1 μM arachidonic acid (Sigma, St. Louis, MO, USA) in RPMI-1640. PGE₂ concentrations in the medium were then determined using an Endogen PGE₂ Competitive ELISA (Endogen, Cambridge, MA, USA), according to the manufacturer's instructions.

Western blotting. Antibodies against COX-2 (Cayman Chemical, AnnArbor, MI, USA), B-cell lymphoma-2 (BCL-2) (Cell Signaling Technology, Danvers, MA, USA), B-cell lymphoma-extra large (BCL-xL) (Cell Signaling Technology), myeloid cell leukaemia-1 (MCL-1) (Cell Signaling Technology), BCL-2 associated X protein (BAX) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology) were used in western blotting. Equal protein loading was evaluated by GAPDH. Cultured cells were collected and lysed with a lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP40, 1 mM EDTA, and a complete protease inhibitor cocktail tablet (cOmplete, Mini Protease Inhibitor Cocktail Tablets; Roche Applied Science, Indianapolis, IN, USA) for 60 min on ice. The extracts were sonicated three times each for 5 s at 20 kHz. The sonicated cell extracts were centrifuged at 20,630 ×g for 30 min at 4°C, and supernatants were collected. Protein concentrations were determined by Protein Assay Rapid Kit Wako (Wako Pure Chemical Industries, Ltd. Osaka, Japan). Equal amounts of protein (50 μg) were mixed with sodium dodecyl sulfate (SDS) sample buffer containing 2-mercaptoethanol, boiled for 5 min, and loaded on 7% SDS-Poly-Acrylamide Gel Electrophoresis (PAGE) gels. Protein was transferred to polyvinylidene difluoride membranes (GE Healthcare, Milwaukee, WI, USA) with a tank transfer system (Bio-Rad, Hercules, CA, USA). The membranes for the antibody reaction were blocked in 5% skimmed milk at room temperature for 1 h. Membranes were reacted with specific primary antibodies diluted as follows: COX-2 1:500, BCL-2 1:1,000, BCL-xL 1:1,000, MCL-1 1:1,000, BAX 1:500, and GAPDH 1:500, at room temperature for 1 h. The bound primary antibody was detected by a secondary horseradish peroxidase-linked appropriate species antibody preparation. Signal detection was performed using an enhanced chemiluminescence (ECL) system (ECL Plus Western Blotting Detection Reagents; GE Healthcare).

Cell proliferation assay. Cell proliferation ability was evaluated by the WST-8 colorimetric assay. COX-2 and control transfectants (1×10³ cells/well) were seeded into 96-well plates in 100 μl CM. After a 96-h incubation, 10 μl of WST-8 reagent solution (Cell Count Reagent SF; Nacalai Tesque Inc.) was added and cells were incubated for 1.5 h. Cell viability was determined by a colorimetric comparison in which optical density values were read from a microplate reader at an absorption wavelength of 450 nm (A₄₅₀).

Chemosensitivity testing. 5FU and cisplatin were generously provided by Kyowa Kirin (Tokyo, Japan) and Nippon Kayaku (Tokyo, Japan), respectively. Cells were plated at 1×10³ cells/ml in 96-well plates in a volume of 100 μl and incubated for 24 h. The media were discarded and cells were incubated in the presence of graded concentrations of 5-FU (0.1, 1, 10, and 100 μg/ml) or cisplatin (0.1, 1, 10, and 100 μg/ml) for 72 h. Cell sensitivity to 5-FU and cisplatin was then evaluated by the WST-8 assay, as described above. The percentage of surviving cells was estimated by dividing the A₄₅₀ of treated cells by the A₄₅₀ of control untreated cells.

In the COX-2 inhibition experiment using siRNAs, transfectants (1×10³ cells/well) were seeded on a 96-well plate in 100 μl CM and siRNA was transiently transfected as described below. After 24 h, the media were discarded. Cells were treated with graded concentrations of 5-FU and cisplatin for 72 h. Cell viability was determined by the WST-8 assay.

COX-2-specific inhibition by siRNAs. siRNA (siD324) specific for COX-2 (GenBank Accession No. NM_000963) was prepared for COX-2 inhibition. siRNA and random siRNA (siD325) for the control were obtained from Takara Bio Incorporated (Otsu, Japan). Sense and anti-sense strands of siRNAs were as follows: (sequence siD324), 5’-CAAACGCUGAUUACAGAUATT-3’, sense, and 3’-TTGUUUGCGACUAAUGUCUAU-5’, antisense; siRNA-random (sequence siD325), 5’-UCUUAAUCGCGUAUAAGGCTT-3’, sense, and 3’-TTAGAAUUAGCGCAUAUUCCG-5’, antisense. All of these sequences were determined through a BLAST search (http://blast.ncbi.nlm.nih.gov/), to avoid sharing sequence homology with any known human mRNA. Transient transfection into COX-2 transfectants was performed using Trans IT-TKO (Invitrogen).

Statistical analysis. Data are reported as the mean±standard deviation. The Student's t-test was used for statistical comparison. Differences were considered significant if a p-value less than 0.05 was obtained.