Human GC cell lines (MKN45, KATOIII, MKN74, MKN28 and MKN1) were obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan). MKN45 and KATOIII cells were derived from poorly-differentiated adenocarcinomas. MKN74 and MKN28 cells were derived from moderately- and well-differentiated adenocarcinomas, respectively. The MKN1 cell line is a gastric adenocarcinoma cell line obtained from a metastatic lymph node and has the ability to differentiate into either adenomatous or squamous cells. The KGC01 line was obtained from a patient with clinically diagnosed GC with a pathological diagnosis of type 4 carcinoma (pT4N1H0CY1M0/stage IV); this patient provided informed consent (approved by the ethics committee of Keio University; no. 17–47). These cell lines were cultured in RPMI-1640 (Sigma, St. Louis, MO) containing 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA) and penicillin-streptomycin mixed solution (penicillin 10,000 units/ml, streptomycin 10,000 μg/ml; Nacalai Tesque, Inc., Kyoto, Japan). In all experiments, cells were cultured at 37˚C in a humidified 5% CO₂/95% air atmosphere. Cetuximab (Erbitux, Merck, Lyon, France) was obtained from Bristol-Myers Squibb Co. (New York, NY), 5FU was obtained from Kyowa Hakko Kirin Co., Ltd. (Tokyo, Japan) and tegafur, gimeracil and oteracil, all of which are components of S-1, were synthesized by Taiho Pharmaceutical.

To detect expression of EGFR, cells were removed from the culture dish using trypsin and EDTA, pelleted by centrifugation, washed with phosphate-buffered saline (PBS) and resuspended at 37˚C in Hanks' balanced salt solution (HBSS) containing 2% FBS and 10 mM HEPES (Invitrogen). Cells were incubated with Blocking One solution (Nacalai Tesque) for 20 min at room temperature. They were then washed and incubated with anti-human EGFR antibody conjugated with FITC (BD Pharmingen, San Jose, CA) for 30 min at 4˚C. Finally, cells were subsequently counterstained with 1 μg/ml propidium iodide (PI; Sigma) to label dead cells. Then, 1×10⁶ viable cells were analyzed using a FACSVantage™ SE (Becton-Dickinson, San Jose, CA). The distribution of cells was analyzed using FlowJo software (Tomy Digital Biology, Tokyo, Japan).

otal RNA from cells was extracted using an RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The concentration of total RNA was determined using a NanoDrop ND-1000 (NanoDrop Technologies, San Diego, CA). Briefly, purified total RNA was reverse-transcribed to generate double-stranded cDNA using Eagle Taq Master Mix with ROX (Roche Applied Science, Indianapolis, IN) and the expression of human EGFR was analyzed using the Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). TaqMan gene expression assay primers and probe mixes were used for GAPDH and EGFR (assay IDs Hs99999905_m1 and Hs01076078_m1, respectively; Applied Biosystems). GAPDH was detected using TaqMan primers and probes and was used as the control gene. The thermal cycling reaction included incubation at 95˚C for 20 sec and 40 cycles of 95˚C for 3 sec and 60˚C for 30 sec. Data were collected using analytical software (Applied Biosystems). Using the ΔΔC_T method, the expression level of each gene was determined relative to the value of the expression of the gene in TMK-1 cells.

DNA was extracted from GC cell lines using a QIAmp DNA Mini kit (Qiagen, Duesseldorf, Germany). A NanoDrop ND-1000 (NanoDrop Technologies) was used to evaluate the concentration of the samples. The reaction mixes contain a single primer set specific for either the wild-type or mutated sequences in codons 12 and 13 of K-ras. Direct sequencing was done using a Big Dye Terminator cycle sequencing kit (Applied Biosystems) and analyzed on an ABI PRISM 310 DNA Analyzer automated sequencer (Applied Biosystems).

Cells were plated in 96-well micro-plates and cultured for 12 h before exposure to various concentrations of compounds for 72 h. Cells were quantified using the WST-8 assay. The optical density (OD) value was detected by Rainbo Sunrise (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The rate of inhibition was calculated as follows: % inhibition = (OD of treated group − blank)/OD of control group × 100. The concentration of tested drugs resulting in 50% growth inhibition (IC₅₀) was calculated. Data were analyzed to determine the combination index (CI), a well-established index of the interaction between two drugs (16). CI values of <1, 1 and >1 indicate synergistic, additive and antagonistic effects, respectively.

To evaluate the dependency of cetuximab activity on EGFR and AKT phosphorylation, cells were exposed to 3.97 μM cetuximab for 72 h before they were collected. Cells were washed in PBS and fixed with 2% paraformaldehyde (PFA) in a 37˚C water bath for 10 min. Then, cells were washed with PBS and pelleted by centrifugation (800 × g) for 5 min and the supernatant was removed. Cells were mixed to disrupt the pellet and permeabilized by adding 500 μl of 90% methanol (for 1–10×10⁶ cells) and incubated on ice for 15 min. After blocking on ice for 10 min, cells were then washed and incubated with primary antibodies against EGFR, AKT, phospho-EGFR (Tyr1068) and phospho-AKT (Ser473) (Cell Signaling Technology, Inc., Danvers, MA) for 60 min at room temperature. Cells were washed with PBS before incubation for 30 min with Alexa Fluor 488 donkey anti-rabbit IgG antibody (Invitrogen). Then, each sample was analyzed using a FACSCalibur™ (Becton-Dickinson). Cell distribution was analyzed using FlowJo software (Tomy Digital Biology).

For apoptosis assays, the supernatant was aspirated and cells were resuspended in 150 μl binding buffer, and stained with 5 μl Annexin V-FITC and 5 μl PI at room temperature for 25 min in the dark. After incubation, cells were processed as directed in the kit instructions (Annexin V-FITC Apoptosis Detection Kit I, BD Pharmingen) and analyzed using a FITC signal detector and PI detector using a flow cytometer (FACSCalibur™) and Cell Quest software (Becton-Dickinson).

MKN28 cells (1×10⁶) were implanted s.c. into the axilla of 6-week-old male athymic nude mice. Drug administration was initiated when tumors in each group achieved an average volume of 333±2.16 mm³. Mice were randomly allocated to control and treatment groups. Treatment groups consisted of control, S-1 alone, cetuximab alone and combination S-1/cetuximab. Each treatment group included 8 mice. S-1 was administered at a dose of 6.9 g/kg/day and given by oral gavage daily for 14 days. Cetuximab (40 mg/kg/day) was given i.p. on Days 1, 4, 8 and 14. Control animals received sterile PBS administration. Tumor volume was determined from caliper measurements of tumor length (L) and width (W) according to the formula LW²/2 and on body weights acquired every 3 days and on the day of evaluation. Both tumor size and body weight were measured three times per week. The percentage of tumor growth inhibition (TGI%) was calculated as follows: TGI (%) = [1 − (tumor volume of treatment group on evaluation day − tumor volume of treatment group on Day 1)/(tumor volume of control group at evaluation day − tumor volume of control group on Day 1)] × 100. The percentage of body weight change (BWC%) was calculated as follows: BWC (%) = [(BW on evaluation day) − (BW on Day 1)]/(BW on Day 1) × 100.

Samples were fixed with 4% PFA for 24 h at RT. Immunohistochemical staining for EGFR was performed on 4-μm thick sections placed on pre-coated slides with APS (Matsunami Glass Ind., Ltd., Osaka, Japan). Briefy, slides were incubated with blocking reagent-N101 (Wako Pure Chemical Industries, Tokyo, Japan) for 20 min. After rinsing in PBST, avidin and biotin blocking was performed for 15 min each. Slides were incubated with anti-human EGFR monoclonal antibody (Cell Signaling Technology, Inc.). EnVision™+, Rabbit/HRP (Dako, Glostrup, Denmark) was then used as secondary antibody for 30 min. DAB staining reactions were conducted for 10 min. Slides were counterstained with haematoxylin. Finally, slides were cover-slipped with aqueous mounting medium (Aquatex^®, Merck). Specimens were analyzed under a light microscope, and EGFR positivity was defined as strong membrane and cytoplasmic staining in at least 50–75% of cells.

All data were expressed as the mean ± SD. Statistically significant differences were determined using two-tailed Student's t-test or χ² test. P-values <0.05 were considered statistically significant.

To evaluate EGFR amplification and K-ras mutation status, six GC cell lines (MKN28, MKN45, MKN74, KATOIII, TMK-1 and KGC01) were examined for their expression of EGFR protein and mRNA by flow cytometry and real-time PCR and for mutation analysis by direct sequencing. FACS analysis revealed that EGFR expression was significantly higher in MKN28 cells compared to other cell lines (P<0.05) (Fig. 1A). The EGFR mRNA expression ratio of each cell line was determined relative to the value of TMK-1. The EGFR mRNA level was 43.08±0.53-fold for MKN28 cells, 24.57±0.62-fold for MKN45 cells, 15.52±2.79-fold for MKN74 cells, 38.19±2.07-fold for KATOIII cells and 4.90±1.12-fold for KGC01 cells (Fig. 1B). K-ras mutation status for each cell line is shown in Table I. Among the GC cell lines examined, EGFR protein and mRNA were overexpressed in MKN28 cells, while KATOIII cells showed amplification of EGFR mRNA, but were negative for EGFR protein expression.

The effects of 5FU or cetuximab monotherapy or combination 5FU/cetuximab therapy on the growth of GC cells with and without EGFR amplification were evaluated. 5FU was used instead of S-1 for these in vitro experiments, since tegafur, a component of S-1, is metabolized to 5FU in the liver. The combined effect of 5FU and cetuximab was evaluated on the basis of the CI. 5FU monotherapy inhibited the proliferation of GC cells, although the IC₅₀ values varied significantly between the individual cell lines (Fig. 2A and B). On the other hand, EGFR-amplified MKN28 cells showed only sensitive to cetuximab in a concentration-dependent manner compared with other GC cells (Fig. 2C). The combination of 5FU and cetuximab exhibited a synergistic inhibitory effect on the growth of EGFR-amplified MKN28 cells (C.I. value = 0.92±0.015), but not on cells without EGFR amplification, including MKN74 and TMK-1 cells (Fig. 2C–F).

EGFR can signal through the AKT or MAPK pathways (17). To explore the anti-proliferation mechanism of EGFR-targeted agents, we examined the effects of cetuximab on the EGFR/AKT signaling pathway. MKN28 and TMK-1 cells were treated with cetuximab for 72 h. In the EGFR-amplified cell line MKN28, cetuximab decreased both EGFR and AKT phosphorylation when compared with the isotype controls. In contrast, phosphorylation of EGFR or AKT was not affected by cetuximab in TMK-1 cells, in which EGFR is not amplified (Fig. 3A). These data indicate that cetuximab can suppress the activation of key pathways that are downstream of EGFR.

To investigate the mechanism underlying the synergistic growth inhibition induced by combination of 5FU and cetuximab, we examined the effects of each agent alone and in combination on apoptosis in GC cells. An assay based on the binding of Annexin V to the cell surface revealed that the frequency of apoptosis was markedly greater in EGFR-amplified cells treated with both 5FU and cetuximab than in cells treated with either agent alone (Fig. 3B). No such effect was observed in cells negative for EGFR amplification. These data indicate that the combination of 5FU and cetuximab exhibits an enhanced apoptotic effect in EGFR-amplified GC cells, but not in those without EGFR amplification.

The antitumor activities of cetuximab combined with chemotherapy were examined in an EGFR-overexpressing human GC xenograft model. Mice with tumors derived from MKN28 cells were divided into groups for treatment with vehicle, S-1, cetuximab, or combined S-1/cetuximab for 14 days. Tumor volume (TV) was evaluated between groups at the end of the experiment. The TV (g) for combined S-1/cetuximab was 0.22±0.05 g, whereas for control, S-1 and cetuximab alone was 20.0±1.96 g, 0.27±0.07 g and 0.30±0.17 g, respectively. Additionally, the TGI % for cetuximab combined with S-1 was 43.2%, while that for S-1 and cetuximab alone was 29.8 and 22.4%, respectively. Combination S-1/cetuximab therapy inhibited the growth of tumors formed by EGFR-amplified MKN28 cells compared to treatment with either agent alone (P<0.05) (Fig. 4A). All treatments were well tolerated by the mice, with no signs of toxicity or weight loss during therapy (Fig. 4B). Furthermore, tumors in each treatment group were examined for expression of EGFR protein by IHC. EGFR expression was decreased in the cetuximab alone and the S-1/cetuximab groups compared to the control and S-1 alone groups (Fig. 4C). Thus, the combination S-1/cetuximab therapy appears to result in an enhanced antitumor effect in EGFR-amplified GC xenografts, consistent with the results obtained in vitro.

Among the 40 specimens, the median age of patients was 67 years (range, 32–94 years); 20 patients had differentiated carcinoma and the other half had undifferentiated carcinoma. Formalin-fixed paraffin-embedded specimens from all 40 patients were examined for EGFR by IHC (Fig. 5). Analysis of EGFR protein expression by microscopic observation revealed a score of 0 in24 cases (60%), 1+ in 5 cases (12.5%), 2+ in 6 cases (15%) and 3+ in 5 cases (12.5%). EGFR reactivity was not correlated with gender, age, differentiation, lymph node metastasis or distant metastasis. Significantly more clinicopathological stage III/IV cases had EGFR-positive disease (30%) compared to stage I/II cases (10%, P=0.0088) (Table II). These results indicate that EGFR positivity is associated with advanced disease in GC.