Introduction 1 2 1 4 5 10 11 12 13 14 13 15 16 In this study, we established a cisplatin-resistant cell line from an OCSS-derived cell line and investigated the differential EGFR and phosphorylated EGFR (p-EGFR) expression between OSCC cell lines and the cisplatin-resistant sublines. In addition, we examined the effect of combination therapy with an EGFR inhibitor and cisplatin on the growth of OSCC cells. Materials and Methods Cell Lines 2 17 18 Cell Growth Analysis with MTT Assay U p Western Blot Analysis 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. Results Single Agent Effects 1 1 Fig. 1 a b Materials and methods  Table 1 Cisplatin and AG1478 sensitivity of OSCC cell lines Cell line IC50 IC50 Cisplatin (μg/ml) AG1478 (μM) H-1 0.49 25.3 Sa-3 0.43 28.5 H-1/CDDP 6.5 23.3 Sa-3/CDDP 5.9 21.2 Combination Effects 2 Fig. 2 p  Expression of EGFR and p-EGFR 3 Fig. 3 Materials and methods  Discussion 14 3 19 20 21 23 22 23 21 16 24 24 26 Augmented effects of inhibition of OSCC cell growth through the combination of AG1478 and cisplatin provide a potential and novel strategy for patients with oral cancer with acquired cisplatin resistance. In addition, it would be very advantageous if an equal chemotherapeutic effect could be obtained with a smaller dosage of cisplatin. The combination of AG1478 and cisplatin against OSCC definitely deserves additional in vivo and clinical studies.