The human lung cancer cells (A549, NCI-H1299 (H1299), NCI-H460 (H460) were cultured in RPMI-1640 (Gibco Life Technologies, Carsbad, CA, USA) medium supplemented with 10% fetal bovine serum (Hyclone, GE Healthcare Life Sciences, Logan, UT, USA) and incubated at 37°C in a humidified air containing 5% CO₂.

Cells were exposed to ionizing radiation at room temperature using Faxitron RX-650 X-rays (Faxitron Bioptics, LLC, USA). The dose rates were 0.765 Gy/min.

siRNA against Nrf2, Notch1 and non-targeting negative control siRNA were purchased from Invitrogen (Invitrogen Life Technologies, Carlsbad, CA, USA). Cells were seeded onto new plates one day prior to transfection. Transfection reagent was performed with Lipofectamine 2000 (Invitrogen Life Technologies), following the manufacturer's protocol. The serum-free medium was replaced with new culture medium for 6 h after transfection.

Total RNA was extracted from cultured cells by using TRIzol reagent (Takara Biotech Co., Ltd.) and reverse transcription was carried out using a PrimeScript RT Master Mix (Takara Biotech Co., Ltd.) in a total volume of 20 μl.

Quantitative PCR was carried out with SYBR Premix Ex Taq II (Takara Biotech Co., Ltd.), 50 ng DNA and 10 μM of each of the following primer pairs: Nrf2, 5′-AGCCCAGCACATCCAGTCA-3′ (forward) and 5′-TGCATGCAGTCATCAAAGTACAAAG-3′ (reverse), Hes1, 5′-GTGTCAACACGACACCGGATAAAC-3′ (forward) and 5′-CAGAATGTCCGCCTTCTCCAG-3′ (reverse), β-actin, 5′-TGGCACCCAGCACAATGAA-3′ (forward) and 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′ (reverse), β-actin for coding genes. The reaction was conducted on an FTC-3000 qPCR system (Shanghai Funglyn Biotech Co., Ltd.), with the following cycling conditions: 95°C for 30 sec, 40 cycles of 95°C for 5 sec and 59°C for 30 sec. The expression of genes of interest was normalized to that of β-actin in all samples. Relative quantification approach (ΔΔCt) was used to calculate the fold change.

Intracellular ROS levels were assessed using 10 μM 2′,7′-dichlorofluorescin diacetate (DCFH-DA; Molecular Probes, Sigma), as described previously (26). Briefly, cells were treated for 30 min at 37°C in the dark. After incubation, cells were gently washed with PBS to remove the dye. Samples were observed with confocal microscopy (LSM700; Carl Zeiss) or harvested by trypsinization, washed, resuspended in 1 ml of PBS, mean intensity was measured at an excitation wavelength of 488 nm and an emission wavelength of 525 nm using multimode reader (Thermo Varioskan Flash 3001).

Cells were washed with cold PBS and lysed with RIPA buffer (Beyotime, Haimen, China) with protease inhibitor cocktail. Cells were harvested for 30 min on ice and collected at 10,000 g for 15 min at 4°C. The total concentrations of samples were measured using BCA protein assay kit (Pierce, Rockford, IL, USA). Equal amounts of protein were loaded onto 10% SDS-PAGE and proteins were transferred to an immobilon PVDF membrane (Roche). The membranes were then blocked with 0.05% Tween and 5% BSA (BBI Life Sciences Corp., Canada) in Tris-buffered saline for 2 h at room temperature and incubated overnight at 4°C with primary antibodies against Nrf2, GCLC, HO-1, NQO1, Bax, Bcl-2, cleaved (active) caspase-3, and PARP-1 cleavage (1:500, Cell Signaling Technology, Danvers, MA, USA). The preparative membranes were reacted with appropriate secondary antibodies conjugated to HRP. The immunological complexes were visualized with electrochemiluminescence (Millipore, Darmstadt, Germany). Band intensities were analyzed by ImageJ software.

Cells were washed twice with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. After washing twice with PBS, fixed cells were stained with Hoechst 33258 and incubated for 10 min in the dark and then washed with PBS. Apoptotic cells were identified by condensation and fragmentation of nuclei examined by fluorescence microscopy. Apoptotic cells were counted using cellprofiler 2.1.1 software (Broad Institute, Cambridge, MA, USA).

Cells were pretreated 24 h with siRNA targeting Nrf2 and then exposed to 4 Gy of X-ray radiation. The medium was aspirated followed by three PBS washes. Using 4% paraformaldehyde, cells were fixed onto cover slips for 30 min at room temperature. Cells were rinsed three times with PBS and incubated with 0.5% Triton X-100 for 10 min. The cells were washed three times and blocked with 0.1% BSA in PBS for 1 h. Primary anti-Nrf2 antibody (Cell Signaling Technology) was added and incubated overnight at 4°C. After three washes with PBS, the cells were incubated for 1 h at room temperature with the appropriate secondary antibody. After PBS washes, the slides were incubated with 0.5 mg/ml DAPI (4′,6′-diamidino-2-phenylindole) at room temperature for 5 min. All images were observed under a confocal microscope equipped with a digital camera (LSM700; Carl Zeiss).

Results are presented as mean ± standard deviation (mean ± SD). The data were analyzed using Student's t-tests. A p-value of <0.05 was considered statistically significant.

Nrf2 is induced by external stimulation (15), such as IR. To investigate the effect of IR on Nrf2, A549 cells were irradiated with varying doses of X-rays radiation. The protein expression levels of Nrf2 were measured by western blot analysis. The results showed that Nrf2 was induced in a dose-dependent manner from 4 to 8 Gy. Furthermore, the expression of Nrf2 was increasingly elevated from 4 to 24 h after a single dose of 4 Gy (Fig. 1A and B). The effective dose was 4 Gy and the maximal effect was observed at 24 h, therefore, we chose these parameters for our further experiments.

A549 cells were treated with non-targeting siRNA or siRNA specific to Nrf2 at 24, 48 and 72. There were no significant changes in expression levels of the Nrf2 in cells transfected with a scrambled siRNA sequence (Fig. 2A). Whereas, both protein and mRNA levels were significantly decreased after being transfected with siRNA targeting Nrf2 (Fig. 2B and C). These results conveyed that the selected siRNA efficiently knocked down the expression of Nrf2, especially at 48 h time-point. Inhibition of Nrf2 by siRNA significantly decreased radiation-induced Nrf2 expression at mRNA levels (Fig. 3A). Nrf2 was localized at both the cytoplasm and the nucleus in control cells, but IR prominently stimulated translocation of Nrf2 into the nucleus. Knockdown of Nrf2 significantly attenuated IR-induced Nrf2 nuclear translocation (Fig. 3B). Taken together, these results indicated that IR induced Nrf2 nuclear localization and this effect could be counteracted by Nrf2 knockdown.

To further confirm the inhibitory effect of Nrf2 knockdown on antioxidant responses in A549 cells, we measured the function of the Nrf2 knockdown (Fig. 4). Notably, IR significantly increased Nrf2 target proteins GCLC, HO-1 and NQO1 expression in A549 lung cancer cells. Whereas, lowering Nrf2 expression levels led to a significantly decreased expression of Nrf2 target proteins GCLC, HO-1 and NQO1. These results showed that RNAi-mediated reduction of Nrf2, at least partly, decreasing the levels of antioxidant proteins induced by IR.

To determine whether the decrease in Nrf2 function increased ROS generation after exposure to IR, intracellular ROS levels were monitored using the fluorescent indicator DCFH-DA at 24 h post irradiation in A549 cells. Knockdown of Nrf2 distinctly increased ROS after X-ray radiation as seen in confocal images and the mean fluorescent intensity (MFI) compared with NC+IR (Fig. 5). To some extent, lowering Nrf2 also effected the generation of ROS. Exposure to IR combined with knockdown of Nrf2 further enhanced the ROS production and increased the MFI compared to unirradiated cells. These results suggested that decreased Nrf2 activity enhanced the generation of ROS after irradiation.

Previous studies have shown cross-talk between Nrf2 and the Notch pathway (27–31). To test whether Notch1 expression could be regulated by Nrf2 after irradiation, we treated A549 and H460 cells, which express Keap1 protein with different domain mutations, and H1299 cells, which express no mutation in Keap1. The expression of Notch1 was upregulated after radiation, while, in Nrf2 knockdown cells, Notch1 was debilitated (Fig. 6A). Furthermore, the expression of Hes1, a downstream gene in the Notch1 pathway (32), was reduced substantially after X-rays radiation in Nrf2 knockdown cells (Fig. 6B). We also demonstrated Notch1 was accumulated in A549 cells, but was reduced in Nrf2 expressing cells after irradiation. Collectively, these findings confirmed that Notch1 activation was abolished by the knockdown of Nrf2 in A549, H460 and H1299 cells.

Bcl-2 family members, caspase-3 and PARP-1 are crucial mediators of apoptosis. To determine whether downregulation of Nrf2 and Notch1 following radiation induce apoptosis, we examined the expression of these mediators. Knockdown of Notch1 induced a sharp increase in the protein level of Bax, cleaved caspase-3 and PARP-1 cleavage and a sharp decrease in the protein level of Bcl-2 after IR in A549 cells (Fig. 7A). Similarly, knockdown of Nrf2 enhanced radiation-induced cleaved caspase-3 and PARP-1 cleavage expression in all three cell lines (Fig. 7B). Furthermore, the nuclear morphological changes were also measured. Apoptotic cells showed a number of common features, such as cell shrinkage, nuclear condensation, and formation of pyknotic bodies of condensed chromatin. Suppression of Nrf2 resulted in a pronounced increase in cellular apoptosis after irradiation compared with NC+IR alone (Fig. 7C). Together, these findings suggest that lowering Nrf2 expression clearly facilitated IR induced apoptosis in NSCLC cell lines.