Lung cancer cell lines A549 and H1299 were maintained in Dulbecco's modified eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) (both from Hyclone, Logan, UT, USA), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified 5% CO₂ incubator. MG132 (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO) and diluted by DMEM to different concentrations. Cells were divided into 4 groups: control group, MG132-treated group, irradiation alone and MG132 plus irradiation group. Briefly, exponentially growing cells were exposed to different doses of ionizing radiation using X-ray linear accelerator (Rad Source, Suwanee, GA, USA) at a fixed dose rate of 1.15 Gy/min. Control cells were defined as 0 Gy group. Cells were treated with 200 µM MG132 and irradiated 6 h later as previously reported (15).

Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded in 96-well plates at a density of 2.5×10³/well 1 day prior to treatment. Then, cells were treated with MG132 or/and irradiation. After treatment, 20 µl of 5 mg/ml MTT solution was added into each well and incubated for 4 h. After the supernatant was removed, 100 µl of DMSO was added, and then placed in a microplate reader to measure OD value. Cell viability rate (VR) was calculated according to the following formula: VR = (OD in observed group/OD in 0 Gy group) × 100%. All assays were repeated 3 times in quintuplicate.

Cells were treated with MG132 for 24 h prior to irradiation. Apoptosis was measured using propidium iodide (PI)/Annexin V double staining (Keygen, Nanjing, China). Apoptotic fractions were measured using flow cytometry (Beckman, USA). The Annexin V⁺/PI⁻ cells are early in the apoptotic process, the Annexin V⁺/PI⁺ cells indicating late apoptosis. The percentage of both kinds of cells was counted.

Cells were harvested at 48 h after treatment with MG132 or 8 Gy irradiation. Cells were then incubated with ice-cold RIPA buffer (50 mM Tris, pH 7.2, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.05% SDS, and 1 mM PMSF) on ice. The supernatants obtained after centrifugation were quantified with an enhanced BCA protein assay kit (Beyotime, Nantong, China). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) through 10% gel and transferred to polyvinylidene fluoride (PVDF) membrane. Membrane was blocked with 5% skim milk in PBS containing 0.1% Tween-20 (PBST) for 1 h at room temperature. Membrane was probed with antibodies against NF-κB, IκBα (pS36) and lamin B1 overnight at 4°C. Antibodies against NF-κB and IκBα (pS36) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Membrane was washed 3 times in PBST and incubated with peroxidase-conjugated mouse or rabbit secondary antibody for 2 h at room temperature. Membrane was washed in PBST, and the detection was done using enhanced chemiluminescence. The analysis of relative protein expression level was performed using Image J software (National Institute of Health, Bethesda, MD, USA).

After irradiation, cells were treated with 400 ng/ml colchicines (Sangon, Shanghai, China) and incubated for 8 h in an incubator. Cells were harvested; 20 min 75 mM KCl low permeability, and fix in methanol and glacial acetic acid (3:1), stained with 10% Giemsa (pH 7.4). About 600 cells were observed in each group.

All animal experiment protocols in this study were carried out in accordance with a protocol approved by the Institutional ethics Committee. Twenty-four nude mice were obtained from the Laboratory Animal Center of Shanghai (Shanghai, China) and divided into 4 groups: i), control group with mock treatment; ii), radiation group (IR) with 2 Gy/day X-ray irradiation for 5 days; iii), MG132 group; and iv), MG132 plus irradiation group. H1299 cells (1×10⁸) were subcutaneously injected into the hind leg near the spine, in nude mice, respectively. The body weight and tumor volume of nude mice were measured at 2 day intervals for 3 weeks. On day 22, the animals were sacrificed and tumors were resected to perform immunohistochemistry (IHC).

The tissue samples were fixed in 10% neutral-buffered formalin and embedded in paraffin. Three-micrometer-thick paraffin sections were deparaffinized, dehydrated, and heat-treated with citrate buffer (pH 6.0) for 15 min as an epitope retrieval protocol. The endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 30 min, and the non-specific binding sites were blocked with 4% skim milk powder for 30 min. After three washes in phosphate-buffered saline, the sections were incubated with a Ki67 antibody (dilution 1:200; Cell Signaling Technology, Boston, MA, USA) overnight at 4°C. The sections were mixed with 2% skim milk powder to reduce non-specific staining, and a biotinylated secondary antibody was added for 30 min. Avidin-biotin-peroxidase complex (Dako LSAB2 system; Dako Co., Carpinteria, CA, USA) was added, and the color was developed using 3–3′-diaminobenzidine. Counterstaining was performed with hematoxylin. All steps were carried out at room temperature. The positive cells were counted by Image J software.

Statistical analysis was performed with the use of SAS 8.0 statistical software. Data are presented as means ± SEM. The Student's t-test was used to measure the difference between the two groups. The differences between more than two groups were tested for significance using one-way analysis of variance. P<0.05 was considered statistically significant.

Previously, we have determined that MG132 at non-toxic dose (200 nM) enhanced the anti-growth and anti-invasion induced by irradiation (15). To assess whether this dose of MG132 modulates the radiosensitivity of A549 and H1299 cells, we analyzed colony formation after various doses of irradiation. As shown in Fig. 1, the survival fraction was significantly decreased in both cells, especially in MG132 plus irradiation group. The mean lethal dose (D₀) of the irradiation group and MG132 plus irradiation group in A549 cell line were 2.8 and 1.95 Gy, respectively. The mean lethal dose (D₀) in H1299 was 3.63 and 2.32 Gy, respectively. Moreover, the quasi-threshold doses (D_q) were 1.91 and 1.70 Gy for A549 cells, and 4.44 and 1.67 Gy for H1299 cells. The sensitizer enhancement ratio (SER) was 1.44 (A549) and 1.56 (H1299) for cells pretreated with MG132, compared to the control cells. Thus, MG132 sensitized A549 and H1299 cells to irradiation.

We next investigated whether reduced clonogenic survival upon combined treatment with MG132 and X-ray irradiation was associated with increased apoptosis. As shown in Fig. 2, 6 or 8 Gy X-ray irradiation obviously induced apoptosis (Annexin V⁺/PI⁻ plus Annexin V⁺/PI⁺ cells) in both A549 and H1299 cell lines. MG132 treatment further enhanced the apoptotic response of cells to 6 or 8 Gy irradiation. In comparasion, there was no significant difference in apoptotic fraction between irradiation and MG132 plus irradiation at 2 or 4 Gy irradiation. Taken together, these results demonstrate that MG132 augments apoptotic cell death of A549 and H1299 cells in response to irradiation.

Dicentric chromosomes ratio was used to detect chromosome aberration in irradiated cells (16). In this study, dicentric chromosome ratio was determined in H1299 cells after irradiation with or without MG132. As shown in Fig. 3, we found that the dicentric chromosome aberration ratio in MG132 plus irradiation group was higher than of irradiation alone group, suggesting that chromosomes were severely damaged and a positive dose-response relationship exists between dicentric chromosome ratio and the irradiation dose.

NF-κB is a crucial transcription factor that regulates the expression of genes linked to apoptosis, survival, proliferation, invasion, metastasis and tumor cell transformation (17,18). NF-κB has been extensively reported to be implicated in cancer cell radio-sensitivity due to their role of transcriptional regulation upon radiation exposure. To further confirm the effect of MG132 on NF-κB activity induced by irradiation, we analyzed the expression level of NF-κB (p65 subunit) in A549 and H1299 cells. As shown in Fig. 4, the expression of NF-κB in the nuclear extracts of the both cell lines were increased after irradiation, while addition of MG132 decreased radiation-induced NF-κB (p65) expression in the nucleus. Phosphorylation of IκBα, which mediates the degradation of IκBα and activates NF-κB, was decreased, suggesting decreased NF-κB expression was attributed to reduced degradation of IκBα.

We next sought to determine the effect of MG132 on H1299 cells by tumor growth in nude mice. Mice were divided into 4 groups: i), control group with mock treatment; ii), radiation group (IR) with 2 Gy/day X-ray irradiation for 5 days; iii), MG132 group; and iv), MG132 plus irradiation group. H1299 cells were subcutaneously innoculated into nude mice. As shown in Fig. 5A, there was no significant difference in the weight of nude mice between groups. As shown in Fig. 5B, mice treated with MG132 alone yielded 19.58% inhibition of tumor volume as the control group. Compared with the control group, the tumor volume of mice treated with radiation alone was reduced by 35.70%. Comparatively, H1299 xenografts that received combined treatment of 10 Gy radiation and MG132 exhibited much smaller tumors compared with mice that received radiation alone (volume reduction of 62.75% in IR + MG132 group). The expression of Ki67 from xenografts was analyzed by IHC. Ki67 expression was observed by a microscope (He; magnification, ×400). As shown in Fig. 6, Ki67-positive cells were significant decreased in MG132 plus irradiation groups compared with control group. Taken together, these results indicate that MG132 can enhance radiosensitivity of H1299 xenografts.