EGFR Inhibitor Enhances Cisplatin Sensitivity of Oral Squamous Cell Carcinoma Cell Lines

Two human OSCC cell lines have been established at Wakayama Medical University, Wakayama, Japan. The H-1 line was derived from a biopsy specimen of moderately differentiated OSCC in the lower gingiva. The Sa-3 line was derived from a biopsy specimen of well-differentiated OSCC in the upper gingiva. Both cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; Equitech-Bio, Kerrville, TX, USA), 100 units/ml penicillin, and 100 μg/ml streptomycin (Gibco BRL, Grand Island, NY, USA) in a highly humidified atmosphere of 5% CO₂ at 37°C.

In accordance with previously described methods [17, 18], the cisplatin (CDDP)-resistant sublines H-1/CDDP and Sa-3/CDDP were established by repeated subculture in the presence of increasing concentrations of cisplatin (Nippon Kayaku Corporation, Tokyo, Japan), from 0.1 μg/ml until cells became fully resistant to cisplatin and could grow exponentially; in each case the final cisplatin concentration was 0.5 μg/ml. The drug-resistant cell lines were passed in drug-free medium, and there was no loss of resistance during the two-month testing period.

Cells were seeded in 96-well plates at 2000 cells per well in DMEM containing 10% FBS. After 24 h, cells were exposed to one of nine concentrations (0.05, 0.1, 0.25, 0.5, 1, 1.25, 2.5, 5 and 10 μg/ml) of cisplatin or five concentrations (1, 5, 10, 20 and 30 μM) of AG1478 (Calbiochem San Diego CA, USA). After cells were incubated with cisplatin or AG1478 for 24 h, medium was changed to drug-free DMEM and cells were incubated for an additional 72 h. Thereafter, the number of cells per well was quantified with a MTT cell growth assay kit (Funakoshi, Tokyo, Japan) according to the manufacturer’s instructions. Briefly, after 10 μl MTT solution was added to each well, the well was incubated for 4 h and scanned at 550–630 nm by a MTP-300 microplate reader (Corona, Tokyo, Japan). Six wells were used for each drug concentration, and the experiment was repeated three times. The 50% inhibitory concentration (IC50) was calculated from the survival curve. In another experiment, to test whether the combination of cisplatin and AG1478 would achieve higher growth inhibition than the single agent at a concentration lower than the IC50, fixed concentrations of each drug were then tested in combination treatment of OSCC cell lines. All cells were exposed to AG1478 for 1 h before cisplatin. The statistical significance of differences was analyzed by applying Mann–Whitney U-test. The level of significance was set at p < 0.05.

Subconfluent cells were scraped from culture dishes, washed twice with phosphate buffered saline (PBS), and suspended in 700 μl Western blot lysis buffer containing 62.5 mM Tris–HCl (ph 6.8), 25% glycerol, and 2% sodium dodecyl sulfate (SDS). Samples were centrifuged at 15,000 rpm for 20 min at 4°C, after which supernatants were collected. After heating at 95°C for 5 min, equal amounts of proteins were separated with the use of 10% SDS-PAGE. After electrophoresis, proteins were transferred to a PVDF membrane in Tris-glycine buffer containing 20% methanol. The membrane was blocked with 3% skim milk containing 0.01% polyoxyethlene sorbitan monolaurate for 60 min, and incubated overnight with the corresponding primary antibodies {a 1:750 dilution of anti-rabbit polyclonal EGFR antibody [Santa Cruz Biotechnology, Santa Cruz, CA, USA], and a 1:750 dilution of anti-goat polyclonal p-EGFR (Tyr 1173) antibody [Santa Cruz Biotechnology]} at 4°C. The membrane was washed three times for 5 min each with PBS containing 0.05% polyoxyethlene sorbitan monolaurate and horseradish peroxidase-conjugated anti-rabbit or anti-goat antibody for 1 h at room temperature, respectively. Protein signals were visualized by enhanced chemiluminescence using ECL Western blotting detection reagents (Amersham, Arlington Heights, IL, USA) for 1 min and exposed to Kodak Biomax XAR film.