Phorbol 12-myristate 13-acetate (PMA), dimethyl sulfoxide (DMSO), propidium iodide (PI), and thapsigargin were purchased from Sigma-Aldrich; Merck Millipore (Darmstadt, Germany). Tunicamycin was purchased from Enzo Life Sciences (Farmingdale, NY, USA). Carbobenzoxy- valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK) was purchased from Peptide Institute, Inc. (Osaka, Japan). Primary antibodies targeting phosphorylated (p-) eIF2α (Ser51; cat. no. 3597), BiP (cat. no. 3177), caspase-3 (cat. no. 9662), poly (ADP-ribose) polymerase (PARP; cat. no. 9532), and β-actin (cat. no. 4967), and secondary anti-rabbit immunoglobulin (Ig) G horseradish peroxidase (HRP)-linked antibody (cat. no. 7074) and anti-mouse IgG HRP-linked antibody (cat. no. 7076) were purchased from Cell Signaling Technology Japan, K.K. (Tokyo, Japan).

Since the THP-1 human acute monocytic leukemia cell line is negative for p53 expression due to a 26 bp deletion beginning at codon 174 of the p53 coding sequence (13), THP-1-derived macrophage-like cells are suitable for exploring apoptosis in macrophages independent of the p53 pathway. THP-1 human acute monocytic leukemia cells were obtained from RIKEN BioResource Center (Tsukuba, Japan). The cells were cultured in Gibco RPMI1640 (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 1% penicillin and streptomycin and 10% heat-inactivated fetal bovine serum (Japan Bio Serum Co., Ltd., Nagoya, Japan) at 37°C in a humidified atmosphere containing 5% CO₂. THP-1-derived macrophages (hereafter termed macrophages) were prepared as previously described (14). THP-1 cells (2×10⁵ cells/ml) were plated in 35-mm dishes with 2 ml of medium containing 100 ng/ml PMA and cultured for 48 h, then the medium was replaced with fresh PMA-free medium, and the macrophage-like cells were used in experiments.

For the treatments, THP-1 cells (2×10⁵ cells/dish) or macrophages were cultured in the presence of either 50 or 500 nM thapsigargin, or 5 or 20 µg/ml tunicamycin for the indicated time periods. Following treatment, cells were harvested by trypsinization followed by centrifugation (300 × g at 4°C for 5 min) for further analysis. Z-VAD-FMK (50 µM), a general caspase inhibitor, was added 1 h prior to thapsigargin treatment. Irradiation (10-Gy) was performed at 1 h following thapsigargin treatment. Since thapsigargin, tunicamycin, and Z-VAD-FMK were dissolved in DMSO, DMSO was used as vehicle control.

X-ray irradiation (150 kVp, 20 mA, 0.5 mm Al, and 0.3-mm Cu filters) was performed using an MBR-1520R-3 X-ray generator (Hitachi, Ltd., Tokyo, Japan) at a distance of 45 cm from the focus, with a dose rate of 1.02–1.05 Gy/min.

Harvested cells were fixed with 70% ethanol overnight at −20°C. Fixed cells were washed with Ca²⁺- and Mg²⁺-free phosphate-buffered saline [PBS(−)] and then treated with RNase A (200 µg/ml) at 37°C for 30 min to hydrolyze RNA. Following treatment, cells were washed with PBS(−) and stained with propidium iodide (PI; 30 µg/ml) for 30 min in the dark. Finally, the cells were filtered with a 40 µm cell strainer (BD Biosciences, Franklin Lakes, NJ, USA) and cell cycle distribution was analyzed by flow cytometry (Cytomics FC500 with CXP software ver. 2; Beckman Coulter, Inc., Brea, CA, USA).

Harvested cells were lysed in 1X Laemmli Sample Buffer (Bio-Rad Laboratories, Inc., Hercules, CA, USA) containing 2.5% 2-mercaptoethanol, by sonication and boiling for 10 min. Protein concentration was determined using the XL-Bradford assay kit (APRO Science, Tokushima, Japan) and a SmartSpec Plus spectrophotometer (Bio-Rad Laboratories, Inc.). The proteins (~5 µg/lane) were separated using 4–20% Mini-PROTEAN TGX precast gels (Bio-Rad Laboratories, Inc.) and were transferred onto polyvinylidene difluoride (PVDF) membranes from Trans-Blot Turbo Mini PVDF Transfer Packs (Bio-Rad Laboratories, Inc.) using the Trans-Blot Turbo transfer system (Bio-Rad Laboratories, Inc.). Membranes were blocked in TBST buffer (10 mM HCl, pH 7.5, 100 mM NaCl, and 0.1% Tween-20) that contained 5% non-fat milk or PVDF Blocking Reagent for Can Get Signal (Toyobo Co., Ltd., Osaka, Japan) at room temperature for 1 h. The membranes were probed with each primary antibody in the TBST buffer containing 5% non-fat skim milk or Can Get Signal immunoreaction enhancer solution 1 (Toyobo Co., Ltd.) overnight at 4°C. The following primary antibodies were used: Anti-p-eIF2α antibody (1:4,000), anti-BIP antibody (1:4,000), anti-caspase-3 antibody (1:3,000), anti-PARP antibody (1:3,000), or anti-actin antibody (1:4,000). The membranes were then incubated with HRP-conjugated secondary antibodies in TBST buffer containing 5% non-fat milk or Can Get Signal immunoreaction enhancer solution 2 (Toyobo Co., Ltd.) for 1 h. The following secondary antibodies were used: HRP-linked anti-rabbit IgG antibody (1:10,000) or HRP-linked anti-mouse IgG antibody (1:10,000). Antigens were visualized using the Clarity enhanced chemiluminescence western blotting substrate (Bio-Rad Laboratories, Inc.). Blot stripping was performed using Stripping Solution (Wako Pure Chemical Industries, Ltd., Osaka, Japan).

Data are presented as the mean ± standard error. Comparisons between the control and experimental groups were performed using a two-sided Student's t-test. P<0.05 was considered to indicate a statistically significant difference. Excel 2010 (Microsoft Corporation, Redmond, WA, USA) with the add-in Statcel 3 software (The Publisher OMS Ltd., Tokyo, Japan) was used to perform statistical analyses.

The effects of ER stress inducers thapsigargin and tunicamycin were investigated in THP-1-derived macrophages. As demonstrated in Fig. 1A, expression levels of the ER stress protein markers p-eIF2α and BiP were visibly increased in macrophages following treatment with thapsigargin and tunicamycin for 24 h, compared with macrophages treated with DMSO vehicle control. This finding suggests that macrophages responded to ER stress. Ionizing radiation has been reported to induce ER stress in cancer cells (15,16), therefore the expression levels of p-eIF2α and BiP were examined in macrophages following X-ray irradiation. No change was observed in p-eIF2α and BiP protein expression in irradiated compared with untreated cells, suggesting that X-ray irradiation does not induce ER stress in macrophages (Fig. 1B).

The effect of X-ray irradiation and ER stress induction on apoptosis was then examined in THP-1 cells and THP-1-derived macrophages, by cell cycle phase distribution analysis using flow cytometry. As demonstrated in Fig. 2A, X-ray irradiation did not increase the % of macrophages in the sub-G1 fraction, which is a hallmark of apoptosis, compared with untreated macrophages. However, when the immature monocytic THP-1 cells were irradiated, a significant increase in the % of cells in the sub-G1 fraction was observed compared to untreated THP-1 cells (Fig. 2A). These data suggest that the differentiation status of macrophages renders them resistant to ionizing radiation. By contrast, treatment with ER stress inducers thapsigargin and tunicamycin significantly induced the % of cells in the sub-G1 fraction in both macrophages and THP-1 cells, compared to their respective untreated cell control group (Fig. 2B and C). Finally, thapsigargin exhibited a more potent effect in inducing apoptosis in macrophages than tunicamycin, as exhibited by markedly higher numbers of macrophages in the sub-G1 fraction following 72 h of thapsigargin treatment compared with 72 h of tunicamycin treatment (Fig. 2B and C).

Caspase-3 is a key effector protease that is responsible for DNA fragmentation during apoptosis. Therefore, the hypothesis that thapsigargin-induced apoptosis may be mediated by caspase-3 activation was further examined. As demonstrated in Fig. 3A, treatment of macrophages with 500 nM thapsigargin induced caspase-3 cleavage, compared with macrophages treated with DMSO vehicle control. Furthermore, increased cleavage of PARP, which is a target of cleaved caspase-3, was observed in thapsigargin-treated macrophages compared with DMSO-treated macrophages (Fig. 3B). Finally, thapsigargin-induced apoptosis in macrophages was demonstrated to be caspase-dependent, as treatment with the pan-caspase inhibitor Z-VAD-FMK effectively prevented PARP cleavage and reduced the % of cells in the sub-G1 fraction in thapsigargin-treated macrophages (Fig. 3B and C). Taken together, these results suggest that thapsigargin induces caspase-mediated apoptosis.

Previous studies have demonstrated that ER stress causes radiosensitization in cancer cells (11,12). Therefore, the hypothesis that combination treatment with thapsigargin and X-ray irradiation may enhance apoptosis in THP-1-derived macrophages was examined. Treatment with thapsigargin for 48 h induced apoptosis in macrophages, however, combination treatment with 10-Gy irradiation did not affect the % of apoptotic cells compared with thapsigargin treatment alone (Fig. 4). Similarly, combination treatment of macrophages with tunicamycin and 10-Gy irradiation had no effect on apoptosis rate compared with treatment with tunicamycin alone (data not shown). These data suggest that there is no synergistic effect of ER inducers and X-ray irradiation on apoptosis of THP-1 derived macrophages.