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Introduction

Lung cancer, the leading cause of cancer related deaths, is divided into small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC). NSCLC accounts for 85% of all lung cancer cases, moreover, ~70% of cases are at an advanced stage with unresectable tumors (1–3). Radiotherapy is routinely used for treatment of lung cancer. Ionizing radiation (IR) damage, which is caused indirectly by radiolysis of intracellular water, leads to formation of ROS. It has been confirmed that ROS plays a main role in the cytotoxic action after IR. Excessive ROS results in oxidative stress that attacks biological macromolecules and leads to cell deaths (4). Cancer cells possess antioxidant systems results in low endogenous ROS levels and protect cells from the cytotoxic effects of radiation. The capacity of cancer cells is superior to those of normal cells, which leads to the radio-resistance of cancer cells and radiotherapy failure (5).

Nrf2 is a crucial transcription factor regulating the expression of numerous antioxidant genes (6–9). Under basal conditions, Nrf2 is located in the cytoplasm, where it is sequestered by its inhibitor Kelch-like ECH-associated protein 1 (Keap1) (10–12). Under oxidative stress, however, oxidative modification of Keap1 allows Nrf2 to release from Keap1, and then Nrf2 translocates into the nucleus. Once in the nucleus, Nrf2 binds the antioxidant response element (ARE) and drives the expression of several downstream genes such as γ-glutamyl cysteine synthetase modifier subunit (GCLm), GCLC, HO-1 and NQO1 (13–16). Both loss of function mutations in Keap1 and gain of function mutation in Nrf2 lead to Nrf2 overexpression. Accumulated clues show that Nrf2 is overexpressed in various types of cancer cells, including lung cancer, esophageal squamous cancer and skin cancer. Constitutive Nrf2 activation has been implicated in the resistance of cancer cells to radiation therapy (10). Nrf2 has been reported to cross-talk with other pathways. Specifically, Nrf2 strongly regulated Notch1 activity. The Notch signaling is involved in proliferation, differentiation and cell decisions (17–20). Numerous studies have demonstrated that Notch signaling plays a critical role in cancer cells (21–24). It has been reported that Notch1 mediates radio-resistance of glioma stem cells and loss of Notch sensitizes those cells to ionizing radiation (25). However, whether Nrf2 mediated Notch1 downregulation plays a role in response to ionizing radiation remains elusive.

Thus, the blocking of antioxidant responses could increase apoptotic death after radiotherapy. This study investigated the mechanism of the knockdown of Nrf2 enhancing radiation-induced apoptosis. We identified that Notch1 is strongly regulated by Nrf2 in response to IR and revealed that Nrf2 enhances radiation-induced apoptosis through downregulating the expression of Notch.

Materials and methods

Cell cultures

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.

Exposure to ionizing radiation

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.

Transfection

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.

RNA isolation and reverse transcription

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.

Real-time fluorescent quantitative PCR

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.

Measurement of ROS generation

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).

Western blot analysis

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.

Hoechst 33258 staining

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).

Immunofluorescence staining

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).

Statistical analysis

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.

Results

Nrf2 is induced by ionizing radiation in A549 cells

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.

Figure 1 Nrf2 is induced in response to ionizing radiation in A549 cells. (A) Various doses of ionizing radiation after 24 h. (B) A single dose of 4 Gy that was analyzed at indicated time-points. β-actin was used as loading control. Results are from 3 experiments.

Knockdown of Nrf2 decreases radiation-upregulated Nrf2 in A549 cells

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.

Figure 2 Downregulation of Nrf2 in A549 cells. (A) Western blot analysis for Nrf2 activation. Cells were harvested 24, 48 and 72 h after transfection. β-actin served as normalization control. (B) Immunoblot showing reduced levels of endogenous Nrf2 protein following transfection with siRNA targeting Nrf2 (siRNA-Nrf2) or negative control (NC). β-actin served as normalization control. (C) Nrf2 mRNA levels were monitored by quantitative RT-PCR at 24, 48 and 72 h. **p<0.01 versus NC group.

Figure 3 IR-induced nuclear translocation of Nrf2 is suppressed by siRNA-Nrf2 in A549 lung cancer cells. (A) mRNA levels of Nrf2 was determined by quantitative RT-PCR, at 24 h after exposing the cells to 4 Gy of X-ray irradiation. (B) Cells were treated with siRNA-Nrf2 before X-irradiation (4 Gy) and incubated for 24 h. After staining with anti-Nrf2 antibody and Alexa Fluor 647-conjugated secondary antibody (red) and DAPI for nuclear staining (blue), cells were visualized under a fluorescence microscope. Data are representative of at least three different experiments. *p<0.05 versus NC+IR group alone.

Suppression of Nrf2 on the redox status

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.

Figure 4 Effect of Nrf2 knockdown on the redox status in A549 cells. Immunoblot detection of GCLC, HO-1 and NQO1 at 24 h after exposure. β-actin was used as reference control. Experiments were repeated three times.

Knockdown of Nrf2 increases ROS accumulation after exposure

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.

Figure 5 Suppression of Nrf2 activity enhances radiation-induced ROS accumulation in A549 cells. Intracellular ROS levels were determined 24 h post-irradiation, (A) by fluorescence microscopy and (B) by a fluorescent microplate reader. *p<0.05 versus NC+IR group alone.

Knockdown of Nrf2 decreased Notch1 expression after IR

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.

Figure 6 Expression of Notch1 and related gene in the knockdown of Nrf2 cells. (A) Quantification of protein expression. Notch1 were normalized by β-actin expression. (B) Hes1 mRNA levels were measured by quantitative RT-PCR. (C) Immunofluorescence staining for Notch1 in A549 cells, Alexa Fluor 647-conjugated secondary antibody (red) and nuclei were counterstained with DAPI (blue). Images were captured by confocal microscopy and merged. *p<0.05 and **p<0.01 versus NC+IR group alone.

Decrease in Nrf2 and Notch1 increases IR-induced apoptosis

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.

Figure 7 Nrf2 and Notch1 ablation potentiates apoptosis after irradiation. (A) A549, cell lysates were subjected to western blotting for the detection of Bax, Bcl-2, cleaved caspase-3 and PARP-1 activation. β-actin expression was utilized for normalization. (B) Western blot analysis for cleaved caspase-3 and PARP-1 activation. (C) The nuclear morphological changes by Hoechst 33258 staining were assessed. Arrows indicate chromatin condensation and nuclear fragmentation (×200 magnification) and the apoptotic rate of cell population. *p<0.05 versus NC+IR group alone.

Discussion

The Keap1-Nrf2 signaling is the main cellular antioxidant system that regulates a broad spectrum of cytoprotective gene expression against oxidative injure, inflammation and apoptosis (33–36). Keap1 negatively regulates Nrf2 activity by promoting proteasomal degradation of Nrf2. Several reports demonstrated that Keap1 is present in somatic mutations leading to aberrant constitutive Nrf2 activation in NSCLC cells (37–39). Activation of the Nrf2 pathway has been reported to mediate the radio-resistance of lung cancer (40). However, Notch1 regulates cell proliferation, invasion and apoptosis and its dysregulation leads to lung cancer initiation (41) and progression (42). Herein, we demonstrated that Nrf2 regulated the Notch signaling of NSCLC cells. The important finding was that Nrf2 and Notch1 promoted lung cancer cells to apoptosis in coordination.

Nrf2 can be stimulated by ionizing radiation. Several reports have shown that Nrf2 can be activated by 137Cs exposure only after five day in MCF7 cell line (40) and was also induced at 0.1 Gy in Raw 264.7 cells (43). Recently, Nrf2 activation was observed within 6 h in H1299 cells (37). Various types of cells affect the activation of Nrf2. In this study, our results showed a dose-dependent increase of Nrf2 expression after irradiation and the minimum radiation dose inducing accumulation of Nrf2 was 4 Gy. The level of Nrf2 increased at 4–24 h and was strongly elevated at 24 h after exposure to 4 Gy X-rays in A549 cells.

It is well accepted that IR enhance cell apoptosis, which is a major type of cell death. IR produces substantial amounts of intracellular ROS. In cancer cells, it is believed that ROS induced genetic instability and is associated with oncogenic transformation (44). However, ROS must be eliminated to some extent, to avoid potential damage to DNA, and various cellular responses, including apoptosis and death. Diehn et al reported that enhanced ROS scavenger results in low ROS levels, and tumor radio-resistance (45). GCLC, HO-1 and NQO1 are Nrf2-regulated genes and protect against oxidative and xenobiotic stress by mopping up ROS (46–49). In our study, inhibition of Nrf2 significantly attenuated IR-induced Nrf2 nuclear translocation and decreased GCLC, HO-1 and NQO1 expression after irradiation. Our results indicated that decreased level of GCLC, HO-1 and NQO1 lead to increased ROS levels through weak antioxidant capacity. In response to external stimuli, a series of events lead to the stabilization of Nrf2 and its translocation into the nucleus, where Nrf2 exerts its function and controls the expression of antioxidants. However, antioxidants could also regulate Nrf2 activation through feedback effects (50). In a Keap1 mutant cell line, Keap1 hardly regulates Nrf2 activation, and Nrf2 protein levels were high. When introducing exogenous siRNAs, Nrf2 is silenced and few Nrf2 activated, which suggest that Nrf2 nuclear translocation is at low level.

Nrf2 can cross-talk with other critical molecular pathways. Notch signaling is a candidate. Notch pathway influences cell fate determination (21,51), and its activation is a mechanism of radiation resistance in breast cancer (52). Knockdown of Notch1 inhibited the self-renewal capacity in glioma cells (53,54), induced apoptosis in NSCLC (41), and significantly increased cell death after X-ray irradiation (25). Interestingly, Notch1 signaling could be regulated by Nrf2 (16,27,28,30). Several reports have showed that Notch1 expression was reduced in Nrf2 knockout cells (27,28). Nrf2 activated Notch1 signaling and regulated the repopulating capacity of hematopoietic stem progenitor cell (55). Thus, we speculated that there exists a mechanism with a different type of Nrf2 expressing lung cancer cells. For example, A549 and H460 cell lines express high level of Nrf2 (16,39), while H1299 express wild-type Keap1 (37). Our results confirmed our speculation that decrease of Nrf2 mitigates the expression of Notch1 in NSCLC. Notch1 dramatically accumulates after exposure to X-rays. Loss of Nrf2 also reduced radiation-induced Notch1 and Hes1 activation. Our results showed that Notch1 ablation or IR activated the expression of cleaved caspase-3, PARP-1 and pro-apoptotic protein Bax, and decreased the expression of anti-apoptotic protein Bcl-2. Knockdown of Notch1 in A549 cells strongly increased the apoptosis proteins after irradiation. This indicated that loss of Notch1 increased irradiation-induced apoptosis. Similarly, lowering Nrf2 expression induced apoptosis after exposure through increased production of ROS (16,56). RNAi-mediated reduction of Nrf2 could effectively promote cleaved caspase-3 and PARP-1 cleavage activation compared with IR alone in NSCLC cells. Nrf2-Notch pathway is a conserved pathway that is designed to allow cells to respond to the challenge of IR. It coincides with substantial research results, ROS, as inducer of Notch signaling, could activate Nrf2 signaling, which might play a dual trigger role in Nrf2-Notch axis (57–59). It is reasonable to suggest that knockdown of Nrf2 induced production of ROS, thus promoted cellular apoptosis. Downregulation of Nrf2 reduced Notch1 expression, Notch1 also facilitated apoptosis. The double regulation impelled cells to undergo apoptosis.

No information exists regarding the role of Notch in Nrf2 knockdown in NSCLC cells under irradiation. Further investigation to examine the association between Nrf2 and Notch1 is required.

In conclusion, we have shown that knockdown of Nrf2 facilitated ROS generation by blocking cellular antioxidant abilities. Suppression of Nrf2 shows weaker expression of Notch1. These synergistic effect promoted IR-induced apoptosis. This study contributes to future strategies against lung cancer.

Acknowledgements

This study was supported by the Key Program of National Natural Science Foundation of China (U1432248) and the National Natural Science Foundation of China (11105203, 11305224).

References