Human lung fibroblast cell line (Wi-38) and Human tongue squamous cell carcinoma (HSC2, HSC3 and HSC4) cell lines were purchased from Cell Bank, RIKEN BioResource Center (Ibaraki, Japan) respectively. These cell lines were cultured in 100-mm culture dish (Becton-Dickinson Labware, Franklin Lakes, NJ, USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific Inc., Waltham, MA, USA), 5% of antibiotic-antimycotic solution (Thermo Fisher Scientific). In all experiments, cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂.

Four-week-old female Balb/c nu/nu athymic nude mice (average weight 15.0 g) were purchased from CLEA Japan, Co., Ltd. (Tokyo, Japan). The mice were provided with sterile water and food ad libitum, and maintained under pathogen-free conditions in accordance with the Guidelines for Animal Experimentation of Yamaguchi University.

Cetuximab was purchased from Merck Serono (Dermstadt, Germany) and PTX was purchased from Wako Pure Chemical Industries (Osaka, Japan).

Untreated Wi-38, HSC2, HSC3 and HSC4 cells were used for the detection of EGFR expression. PTX and/or cetuximab treated (0, 3, 6, 12, 24 and 48 h) HSC2, HSC3 and HSC4 cells were used for the analysis of p65 NF-κB expression. Whole cell lysates from control and treated cells were prepared with RIPA buffer (Thermo Fisher Scientific) and the amount of protein in the cell lysates were quantified with NanoDrop 1000 (Thermo Fisher Scientific). Cell lysates containing 50 μg protein/sample were subjected to electrophoresis on 10% SDS-polyacrylamide gels, and then transferred to a PVDF membrane. The membranes were blocked and treated with the anti-EGFR rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-p65 NF-κB rabbit polyclonal antibody (Santa Cruz). All antibodies were detected using Western Breeze chromogenic immunodetection system (Thermo Fisher Scientific) according to the manufacturer’s instructions. Anti-actin monoclonal antibody (Santa Cruz) was used for normalization of western blot analysis.

Wi-38, HSC2, HSC3 and HSC4 cells (5×10³ cells/well) were seeded on 96-well plates (Becton-Dickinson Labware) in DMEM supplemented with 10% FBS. Twenty-four hours later, the cells were treated with different concentrations of cetuximab (0, 0.1, 1 and 10 μg/ml) or PTX (0, 0.01, 0.02 and 0.05 μg/ml) alone or in combination to determine the suitable concentration of these drugs in cell growth inhibition. After 48 h, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, 25 μl/well) was added to the 96-well plate and incubated for 4 h. The blue dye taken up by cells was dissolved in dimethyl sulfoxide (100 μl/well), and the absorbance was measured with a spectrophotometer (Bio-Rad Laboratories, Hercules, CA, USA) at 490 nm. Growth inhibitory effects were compared among the groups. All assays were run in triplicate.

Cells (5×10⁵ cells/well) were cultured on cover glasses in 6-well plates (Becton-Dickinson Labware) in DMEM with 10% FBS alone or with PTX and/or cetuximab. Forty-eight hours later, cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde and stained with 2 μg/ml Hoechst 33258 (Dojindo Laboratories, Kumamoto, Japan), a fluorescent DNA binding dye, for 30 min at 37°C. Fluorescence of Hoechst 33258 was observed under a fluorescent microscope (FLoid^® Cell Imaging Station, Thermo Fisher Scientific). Apoptotic cells were identified by their typical morphological appearance, including chromatin condensation, nuclear fragmentation, formation of membrane blebs and apoptotic bodies. The mean percentages of apoptotic cells were estimated by counting 500 cells/area from at least three different areas/sample.

HSC2 cells (1×10⁶) were suspended in 0.1 ml of serum-free medium and injected into the subcutaneous tissue of 5-week old nude mice using a 27-gauge needle. When the estimated tumor volume (0.5 × length × width²) reached 100–150 mm³, the tumor-bearing mice were allocated randomly into control and treatment groups (5 mice/group). HSC2 tumors were treated for 3 weeks with PTX (20 mg/kg, twice/week) and/or cetuximab (20 mg/kg, twice/week) dissolved in 0.5% hydroxypropyl methylcellulose (HPMC). Control group received HPMC (0.5%) only. Tumor size and body weight were monitored and measured every three days. Antitumor effects and body weight changes were compared among the groups. All mice were sacrificed at the end of 3 weeks/21 days.

HSC2 tumors harvested at autopsy were embedded in paraffin blocks. Four-micrometer-thick sections were prepared from the blocks and mounted on slides. These sections were processed for immunostaining using the anti-p65 NF-κB rabbit polyclonal antibody (Santa Cruz), and appropriate peroxidase conjugated anti-rabbit IgG second antibody. Negative controls were done using PBS instead of the primary antibody. The blocking and immunostaining were performed using Dako Envision kit/HRP (Dako, Glostrup, Denmark). Slides were counterstained with hematoxylin. The slides were then examined under a bright-field microscope. A positive reaction was detected as reddish-brown precipitates.

To detect apoptotic cells in mouse tumor tissues, TUNEL assay using the Apoptosis In Situ Detection kit (Wako) was performed, labeling 3′-OH DNA ends generated by DNA fragmentation. Four-micrometer-thick paraffin sections of tumor were deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol. Tissue sections were incubated in 20 μg/ml proteinase K (Dako) for 15 min. After sections were rinsed in distilled water, endogenous peroxidase was blocked by incubating the slides in 3% hydrogen peroxide in PBS for 5 min. After washing with PBS, the sections were incubated with equilibration buffer and then TdT enzyme in a humidified chamber at 37°C for 60 min. They were subsequently put into prewarmed stop wash buffer for 10 min. After rinsing in PBS, the sections were incubated with antidigoxigenin-peroxidase conjugate for 30 min. Peroxidase activity in each section was demonstrated by the application of diaminobenzidine (Peroxidase Substrate kit; Vector Laboratories, Burlingame, CA, USA). Hematoxylin was used as a counterstain. At least 1,000 cells were counted under a microscope in several random fields of each section. The number of apoptotic cells was divided by the total number of cells counted and the result was expressed as a percentage.

All data are expressed as mean ± SD. The significance of the experiment results was determined by the Mann-Whitney’s U test or one-way ANOVA. The differences were considered statistically significant when P<0.05.

To evaluate the expression pattern of EGFR in Wi-38, HSC2, HSC3 and HSC4 cells, western blotting was performed. Wi-38 cells showed relatively weak expression of EGFR compared to the other three cell lines. HSC2 cells showed high EGFR expression compared to HSC3 and HSC4 cells (Fig. 1).

The growth inhibitory effect of PTX and cetuximab on Wi-38, HSC2, HSC3 and HSC4 cells was analyzed by the MTT assay. Cells were treated with different concentrations of PTX (0.01, 0.02 and 0.05 μg/ml) or cetuximab (0.1, 1 and 10 μg/ml) alone for 48 h. PTX inhibited the growth of Wi-38, HSC2, HSC3 and HSC4 cells in a dose-dependent manner (Fig. 2B). Similar result was observed after cetuximab treatment in all cells except Wi-38 cell (Fig. 2A). As combination treatment, we selected the concentrations of 0.02 μg/ml PTX and 1 μg/ml cetuximab as these concentrations significantly inhibited the growth of all cell lines (Fig. 2A and B). As shown in Fig. 2C, 48-h treatment with PTX and cetuximab combination significantly inhibited the growth of HSC2, HSC3 and HSC4 cells compared to PTX or cetuximab alone, or the untreated control.

To understand whether the enhanced cell growth inhibitory effect of PTX and cetuximab combined treatment was due to apoptosis, we performed Hoechst staining to detect DNA fragmentation and chromatin condensation in treated cells. The numbers of apoptotic cells were significantly increased after PTX and cetuximab combined treatment compared to treatment with either agent alone (Fig. 3).

To clarify the mechanisms behind the apoptosis inducing activities of cetuximab and PTX combination treatment, we examined the expression of p65 NF-κB in cells by western blotting. Cetuximab reduced the expression of p65 in a time-dependent manner, while the highest reduction of p65 expression was observed after 12 h of treatment. On the other hand, PTX treatment induced the expression of p65 after 6 h of treatment. Combined treatment with PTX and cetuximab induced p65 expression slightly, and reduced it after 24 h of treatment (Fig. 4).

Nude mice with HSC2 tumor xenografts were used to examine the antitumor activity of PTX and cetuximab single/combination treatment. Control group received 0.5% HPMC only, while treatment groups were treated with either PTX (20 mg/kg/day, twice/week) or cetuximab (20 mg/kg/day, twice/week) alone, or in combination for 3 weeks. Fig. 5 shows the result of the in vivo experiment. All the treatment groups significantly inhibited tumor growth compared to the untreated control. Antitumor effect of cetuximab alone was in the same range as PTX alone. However, the maximum reduction of tumor growth was observed with PTX and cetuximab combination treatment, which is significantly different than treatment with either agent alone. Compared to the control, mice in all treatment groups showed no toxicity or significant weight loss during the treatment.

We examined the expression of p65 in mouse tumors by immunohistochemistry. The expression of p65 was detected in both the nucleus and the cytoplasm of untreated HSC2 tumor cells. However, p65 expression was mainly detected in cytoplasm of cetuximab treated tumors, while it was detected in the nucleus of PTX treated tumor cells. Moreover, the expression of p65 was decreased and was mainly detected in the cytoplasm of tumors that received PTX and cetuximab combined treatment (Fig. 6).

To detect the degree of apoptosis induced by PTX and/or cetuximab in vivo, tumors were removed from mice after treatment and the number of apoptotic cells was quantified by the TUNEL assay. Although treatment with PTX or cetuximab alone moderately induced apoptosis in mice tumors compared to the untreated control, PTX and cetuximab combined treatment significantly upregulated the expressions of TUNEL-positive cells in mice tumors than all other treatment groups or control (Fig. 7).