Phospho-specific antibodies against p38 (Thr-180, Tyr-182; rabbit anti-human; cat. no., 9211), ERK (Thr-202, Tyr-204; rabbit anti-human; cat. no. 9101), and EGFR (Tyr-1068, Thr-669 and Ser-1046/1047; mouse anti-human; cat. nos. 2236, 3056 and 2238, respectively) as well as antibodies against poly-(adenosine diphosphate-ribose) polymerase 1 (PARP-1; rabbit anti-human; cat. no. 9542) and caspase-3 (rabbit anti-human; cat. no. 9662) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibodies against EGFR (1005; rabbit anti-human; cat. no. sc-03) and β-actin (C-11; goat anti-human; cat. no. sc-1615) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). All antibodies were polyclonal, with the exception of EGFR (Tyr-1068) that was monoclonal. Primary antibodies were used at 1:1,000 dilution. Recombinant human TRAIL Apo II ligand was obtained from PeproTech, Inc. (Rocky Hill, NJ, USA). SB203580, U0126 and PD153035, which are inhibitors of p38, ERK and EGFR tyrosine kinase, respectively, were obtained from Merck Biosciences (Danvers, MA, USA), while 5Z-7-oxozeaenol, a selective TAK1 inhibitor, was obtained from Chugai Pharmaceutical Co., Ltd. (Tokyo, Japan). BBR chloride was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All chemical inhibitors, in addition to, BBR chloride were dissolved in dimethyl sulfoxide (Wako Pure Chemical Industries, Ltd.).

MDA-MB-468 cells (ATCC, Rockville, MD, USA) were maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (Invitrogen Life Technologies), 2 mM glutamine (Life Technologies, Gaithersburg, MD, USA), 100 U/ml penicillin and 100 µg/ml streptomycin (Life Technologies) at 37°C in a humidified atmosphere of 5% CO₂. PC-9 cells were provided by Dr Kiura (Okayama University, Okayama, Japan) and were maintained in RPMI-1640 medium (Invitrogen Life Technologies), with identical supplements and under identical conditions.

A WST-1 Cell Counting kit (Dojindo, Kumamoto, Japan) was used to detect cell viability. In brief, cell suspensions in DMEM supplemented with 10% fetal calf serum were seeded into a 96-well plate (5–8×10³/70 µl/well) and incubated at 37°C in a humidified atmosphere of 5% CO₂. Following 24 h of incubation, 10 µl FBS-supplemented DMEM containing the inhibitors at specified concentrations were added to the wells. After 30 min, 10 µl medium containing BBR (Wako Pure Chemical Industries, Ltd.) was added to the respective wells and incubated at 37°C for 90 min, followed by 10 µl medium containing TRAIL (PeproTech, Inc., Rocky Hill, NJ, USA). Plates were then incubated for 24 h at 37°C. WST-1 solution (10 µl) was added to each well 2 h prior to the end of the experiment. Absorbance was measured at 450 nm using a microplate reader (Multiskan Plus Model 355 Microplate Reader; Thermo Labsystems, Helsinki, Finland). Cell viability was determined from the absorbance of soluble formazan dye generated by living cells.

Duplex siRNAs were synthesized at Hokkaido System Science Co., Ltd. (Sapporo, Japan). The target sequences were as follows: p38α, 5′-GCAUUACAACCAGACAGUUGAUAUU-3′; and firefly luciferase (GL2), 5′-CGUACGCGGAAUACUUCGA-3′. MDA-MB-468 cells were transfected with siRNAs in a final concentration of 50 nM using lipofectamine (Life Technologies). At 72 h post-transfection, the cells were treated.

Following stimulation, whole-cell lysates were prepared as described previously (16). Cell lysates were resolved by 7.5, 10 or 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to an Immobilon-P nylon membranes (EMD Millipore, Billerica, MA, USA). The membranes were treated with BlockAce (Dainippon Pharmaceutical Co., Ltd., Suita, Japan) and probed with primary antibodies (dilution, 1:1,000). The antibodies were then detected using polyclonal goat anti-rabbit (cat. no. P0448), polyclonal rabbit anti-mouse (cat. no. P0260) or polyclonal rabbit anti-goat (cat. no. P0449) horseradish peroxidase-conjugated immunoglobulin G antibody (dilution, 1:2,000; Dako, Carpinteria, CA, USA) and visualized using an enhanced chemiluminescence system (Amersham Biosciences, Piscataway, NJ, USA). Certain antibody reactions were performed using in Can Get Signal® immunostain solution (Toyobo Co., Ltd, Osaka, Japan).

Data are presented as the mean ± standard deviation of 3 experiments. The statistical analysis was performed using JMP software (version 10; SAS Institute Inc., Cary, NC, USA). The statistical significance was determined by applying the Bonferroni method (compared to BBR/TRAIL). P<0.05 was considered to indicate a statistically significant difference.

In light of the previous findings of BBR and TRAIL studies (1,17), the present study aimed to investigate the effect of the BBR/TRAIL combination therapy on MAPK activation in EGFR-overexpressing breast cancer cells. The results demonstrated that TRAIL alone markedly upregulated ERK phosphorylation and slightly upregulated p38 phosphorylation in MDA-MB-468 cells (Fig. 1). This effect was not associated with any changes in apoptosis, which may be due to the resistance of MDA-MB-468 cells to TRAIL (Fig. 1, lane 2). By contrast, BBR decreased ERK phosphorylation and markedly increased p38 phosphorylation (Fig. 1). In addition, the BBR/TRAIL combination further enhanced p38 phosphorylation and decreased ERK phosphorylation. TAK1 inhibition using the specific inhibitor 5Z-7-oxozeaenol demonstrated an inhibition of the combination therapy-mediated upregulation of p38; comparably, decreased ERK phosphorylation was observed following 5Z-7-oxozeaenol treatment. This indicated that TAK1 was involved in the regulation of the phosphorylation of p38 and ERK in MDA-MB-468 cells. However, TAK1 inhibition did not induce any changes in combination therapy-mediated apoptosis (Fig. 1, lane 5 vs. 4). In order to further demonstrate the effect of p38 and ERK inhibition on cell apoptosis, cells were pretreated with specific inhibitors, SB203580 and U0126, respectively. The results revealed that p38 inhibition enhanced apoptosis in cells treated with BBR/TRAIL combination therapy, whereas ERK inhibition did not exhibit notable effects on apoptosis (Fig. 1).

In order to confirm the efficient inhibition of p38 in EGFR-expressing cells, the previous experiment was repeated with EGFR tyrosine kinase inhibition instead of TAK1 inhibition. EGFR tyrosine kinase is well-known to be crucial for cell survival and proliferation (18). Although blocking the EGFR tyrosine kinase may indirectly block ERK and EGFR threonine phosphorylation, the present results demonstrated that p38 inhibition downregulated EGFR serine phosphorylation as well as enhanced apoptosis compared with that of EGFR tyrosine kinase inhibition. In addition, ERK inhibition did not promote apoptosis in MDA-MB-468 cells (Fig. 2A). This confirmed that p38, but not ERK, may contribute to cell apoptosis in EGFR-expressing MDA-MB-468 cells. In order to confirm these results, the effects of the EGFR tyrosine kinase inhibitor and p38 inhibitor were examined in EGFR-mutant non-small-cell lung carcinoma (NSCLC) PC-9 cells, the results of which demonstrated a comparable pattern to that of the EGFR-expressing breast cancer cells (Fig. 2B). The consequences of enhancing cellular apoptosis following combination therapy were demonstrated using cell viability assays in these EGFR-overexpressing cell lines; MDA-MB-468 and PC-9. In this experiment, cells were exposed to a lower concentration of BBR, in addition to TRAIL, and the specific inhibitors for extended time periods in order to monitor their significance on cell viability. As shown in Fig. 3A and B, only p38 inhibition in addition to BBR/TRAIL therapy induced a significant decrease in cell viability, compared to ERK inhibition or EGFR inhibition. In addition, the specificity of p38 action was confirmed using specific knockdown of p38 with siRNA. The results demonstrated that p38 knockdown induced EGFR serine downregulation, which resulted in the cleavage of caspase-substrate, PARP-1 (Fig. 3C); this therefore confirmed the value of using p38 inhibition in conjunction with BBR/TRAIL combination therapy in EGFR-expressing cancer cells.