Nurr1 promotes lung cancer apoptosis via enhancing mitochondrial stress and p53-Drp1 pathway

2.1 Cell culture and treatments

In the present study, the BEAS-2B cell line (American Type Culture Collection (ATCC)® no. CRL-9609™), a normal pulmonary epithelial cell line, and the A549 cell line (ATCC® no. CCL-185EMT™), a human lung cancer cell line, were used to determine the influence of Nurr1 on cancer cell viability [28]. The cells were cultured in RPMI-1640 media (Invitrogen, Carlsbad, CA, USA) with 10 % (v/v) fetal bovine serum (HyClone, Logan, UT, USA) at 37°C in a 5 % CO₂ environment. To evaluate the influence of the p53 signaling pathway on A549 cell viability, the pathway blocker pifithrin-α (PFTα) (10 μM, Selleck, Catalog No. S2929) was used for 2 hours at room temperature to prevent p53 activation [29].

2.2 Western blotting

The primary antibodies used in the present study were as follows [30]: Drp1 (1:1000, Abcam, #ab56788), Fis1 (1:1000, Abcam, #ab71498), Opa1 (1:1000, Abcam, #ab42364), Mfn1 (1:1000, Abcam, #ab57602), Mfn2 (1:1000, Abcam, #ab56889), Mff (1:1000, Cell Signaling Technology, #86668), Bcl2 (1:1000, Cell Signaling Technology, #3498), Bax (1:1000, Cell Signaling Technology, #2772), caspase9 (1:1000, Cell Signaling Technology, #9504), survivin (1:1000, Cell Signaling Technology, #2808), p53 (1:1000, Cell Signaling Technology, #9282), complex III subunit core (CIII-core2, 1:1000, Invitrogen, #459220), complex II (CII-30, 1:1000, Abcam, #ab110410), complex IV subunit II (CIV-II, 1:1000, Abcam, #ab110268), Tom20 (1:1,000, Abcam, #ab186735) [31].

2.3 Immunofluorescence microscopy

Cells were washed twice with PBS, permeabilized in 0.1% Triton X-100 overnight at 4°C. After the fixation procedure, the sections were cryoprotected in a PBS solution supplemented with 0.9 mol/l of sucrose overnight at 4°C [32]. The primary antibodies used in the present study were as follows: Tom20 (1:1,000, Abcam, #ab186735), p53 (1:1000, Cell Signaling Technology, #9282), Drp1 (1:1000, Abcam, #ab56788), cyt-c (1:1,000; Abcam; #ab90529).

2.4 TUNEL staining and MTT assay

Apoptotic cells were detected with an In Situ Cell Death Detection Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA; Catalog No. C1024) according to the manufacturer’s protocol [33]. Briefly, cells were fixed with 4% paraformaldehyde at 37°C for 15 min. Blocking buffer (3% H₂O₂ in CH₃OH) was added to the wells, and then cells were permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice. The cells were incubated with TUNEL reaction mixture for 1 h at 37°C. DAPI (Sigma-Aldrich, St. Louis, MO, USA) was used to counterstain the nuclei, and the numbers of TUNEL-positive cells were recorded [34]. MTT was used to analyze the cellular viability. Cells (1x10⁶ cells/well) were cultured on a 96-well plate at 37°C with 5% CO₂. Then, 40 μl of MTT solution (2 mg/ml; Sigma-Aldrich) was added to the medium for 4 h at 37°C with 5% CO₂. Subsequently, the cell medium was discarded, and 80 μl of DMSO was added to the wells for 1 h at 37°C with 5% CO₂ in the dark. The OD of each well was observed at A490 nm via a spectrophotometer (Epoch 2; BioTek Instruments, Inc., Winooski, VT, USA) [35].

2.5 EdU incorporation assay

EdU staining was conducted using the BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 594 (Beyotime, Cat. No: C00788L). Cells were washed with PBS. Fresh DMEM was added, and then, 10 μM EdU was added into the medium. The cells were incubated for 2 hours at 37°C/5% CO₂. After the incubation, the cells were washed with PBS to remove the DMEM and the free EdU probe [36]. The cells were then fixed in 4% paraformaldehyde at room temperature for 30 minutes before being stained with DAPI for 3 minutes. After an additional wash in PBS, the cells were observed under an inverted microscope [37].

2.6 Mitochondrial ROS analysis

Flow cytometry was applied as a quantitative method for evaluating mitochondrial ROS levels according to a previous study. Cells were seeded onto 6-well plates [38]. Subsequently, the cells were isolated using 0.25% trypsin and then incubated with MitoSOX red mitochondrial superoxide indicator (Molecular Probes, USA) for 30 min in the dark at 37°C. Subsequently, PBS was used to wash cell two times, and then the cells were analyzed with a FACS Calibur Flow cytometer. Data were analyzed by FACS Diva software. The experiment was repeated three times to improve the accuracy [39].

2.7 Mitochondrial membrane potential (MMP) evaluation

To observe the mitochondrial potential, JC-1 staining (Thermo Fisher Scientific Inc., Waltham, MA, USA; Catalog No. M34152) was used [40]. Then, 10 mg/ml JC-1 was added to the medium for 10 minutes at 37°C in the dark to label the mitochondria. Normal mitochondrial potential showed red fluorescence, and damaged mitochondrial potential showed green fluorescence. The mPTP opening was measured via tetramethylrhodamine ethyl ester fluorescence according to a recent study [41].

2.8 ELISA

To analyze changes in caspase-9, caspase-9 activity kits (Beyotime Institute of Biotechnology, China; Catalog No. C1158) were used according to the manufacturer’s protocol. In brief, to measure caspase-9 activity, 5 μl of LEHD-p-NA substrate (4 mM, 200 μM final concentration) was added to the samples for 1 hour at 37°C [42]. Then, the absorbance at 400 nm was recorded via a microplate reader to reflect the caspase-3 and caspase-9 activities. To analyze caspase-3 activity, 5 μL of DEVD-p-NA substrate (4 mM, 200 μM final concentration) was added to the samples for 2 hours at 37°C. The levels of antioxidant factors, including GPX, SOD, and GSH, were measured with ELISA kits purchased from the Beyotime Institute of Biotechnology [43]. The experiments were performed in triplicate and repeated three times with similar results.

2.9 Nurr1 adenovirus transfection

Adenovirus-Nurr1 was transfected into cells to perform the gain-of-function assay. The pCMV6-Kan/Neo Nurr1 plasmids were obtained from OriGene Technologies, Inc. Then, 3.0 μg of the above plasmids were transfected into 293 T cells (2×10⁴ cells/well, National Infrastructure of Cell Line Resource) in DMEM with 10% FBS using Lipofectamine® 2000 (Invitrogen) [44]. After 48 hours, the supernatant was collected to obtain the Nurr1 adenovirus (Ad-Nurr1), which was transfected into A549 cells in Opti-MEM media supplemented with Lipofectamine® 2000 according to the manufacturer’s protocol. Transfection was carried out for 48 hours under 5% CO₂ at 37°C. Then, Western blotting was performed to verify the transfection efficiency. Null adenovirus (Ad-ctrl) transfection was used as the negative control group [45].

2.10 Statistical analysis

Data are presented as the means ± S.E.M., and differences were deemed significant when P < 0.05. Data were analyzed using student t test for comparison between two groups, one-way ANOVA for comparison among three or more groups, and non-parameter comparative methods if needed.

3.1 Overexpression of Nurr1 induces A549 lung cancer cell apoptosis and proliferation arrest

To understand the regulatory role of Nurr1 in A549 lung cancer, qPCR and western blotting were used to determine the expression of Nurr1. As shown in Fig. 1A, compared to that in BEAS-2B normal pulmonary epithelial cells, the transcription of Nurr1 was downregulated in A549 cancer cells. In addition, Nurr1 expression was also reduced in A549 cancer cells compared to BEAS-2B normal pulmonary epithelial cells (Fig. 1B-C). These data suggest that Nurr1 is downregulated when normal epithelial cells differentiate into cancer cells. To explore the functional role of Nurr1 in the phenotypes of A549 lung cancer cells, Nurr1-loaded adenovirus (Ad-Nurr1) and control adenovirus (Ad-Ctrl) were transfected into A549 lung cancer cells. The overexpression efficiency was confirmed via western blotting, as shown in Fig. 1D-E. Subsequently, the viability of A549 cells transfected with Ad-Nurr1 was monitored. Compared to transfection with Ad-Ctrl, Ad-Nurr1 transfection reduced cell viability, as assessed via MTT assay (Fig. 1F). Similar results were also obtained in an LDH release assay. As shown in Fig. 1G, compared to Ad-Ctrl transfection, Ad-Nurr1 transfection evoked more LDH release into the medium, indicating that Nurr1 overexpression may promote cell death. This finding was further supported in an analysis of the activity of caspase-3. Compared to that in the Ad-Ctrl group, the activity of caspase-3 was significantly increased in A549 cells transfected with Ad-Nurr1 (Fig. 1H), reconfirming that Nurr1 overexpression triggered A549 death via apoptosis. In addition to cell death, proliferation was also evaluated using EdU staining. As shown in Fig. 1I-J, compared to the Ad-Ctrl group, the Ad-Nurr1 group had a lower percentage of EdU-positive cells, suggesting that Nurr1 overexpression impaired A549 cell proliferation.

Figure 1

Nurr1 is downregulated in A549 lung cancer cells, and overexpression of Nurr1 promotes A549 cancer cell apoptosis. A. qPCR was used to analyze the transcription of Nurr1 in A549 lung cancer cells and BEAS-2B normal pulmonary epithelial cells. B-C. Proteins were isolated from cells, and the expression of Nurr1 in A549 cells and BEAS-2B cells was determined via western blotting. D-E. Nurr1-loaded adenovirus (Ad-Nurr1) and control adenovirus (Ad-Ctrl) were transfected into A549 cells. Then, western blotting was conducted to confirm the overexpression of Nurr1 mediated by Ad-Nurr1 in A549 cells. F. An MTT assay was used to observe alterations in A549 cell viability in response to Nurr1 overexpression. Nurr1-loaded adenovirus (Ad-Nurr1) and control adenovirus (Ad-Ctrl) were transfected into A549 cells. G. Results of an LDH release assay for A549 cells transfected with Ad-Nurr1 or Ad-Ctrl. H. Caspase-3 activity was measured in A549 cells. Nurr1-loaded adenovirus (Ad-Nurr1) and control adenovirus (Ad-Ctrl) were transfected into A549 cells. I-L. EdU staining was used to observe the influence of Nurr1 overexpression on cell proliferation. The number of EdU-positive cells was recorded. *p＜0.05.

3.2 Nurr1 overexpression triggers mitochondrial bioenergetic dysfunction

Next, experiments were performed to observe alterations in mitochondrial bioenergetics in A549 cells transfected with Ad-Nurr1. First, cell ATP production was measured to reflect mitochondrial energy production. Compared to Ad-Ctrl transfection, Ad-Nurr1 transfection reduced ATP production in A549 cells (Fig. 2A). Notably, mitochondria have the ability to generate ATP with the help of the mitochondrial respiratory complex [46, 47]. Accordingly, western blotting was used to observe changes in the expression of the mitochondrial respiratory complex. Compared to Ad-Ctrl transfection, Ad-Nurr1 transfection drastically reduced the levels of the mitochondrial respiratory complex (Fig. 2B-E), indicating that Nurr1 overexpression triggered mitochondrial respiratory dysfunction. In addition, cell energy metabolism is highly dependent on mitochondrial membrane potential [30]. Accordingly, a JC-1 probe was used to detect alterations in mitochondrial membrane potential. As shown in Fig. 2F-G, normal A549 cells exhibited more JC-1 red fluorescence than green fluorescence, indicative of high mitochondrial membrane potential. Interestingly, transfection of Ad-Nurr1 significantly reduced mitochondrial membrane potential, as evidenced by decreased red fluorescence and increased green fluorescence.

Figure 2

Nurr1 modulated mitochondrial bioenergetic dysfunction. A. ATP production was determined in A549 cells. Nurr1-loaded adenovirus (Ad-Nurr1) and control adenovirus (Ad-Ctrl) were transfected into A549 cells. B-E. Western blotting was used to observe alterations in the mitochondrial respiratory complex. Nurr1-loaded adenovirus (Ad-Nurr1) and control adenovirus (Ad-Ctrl) were transfected into A549 cells. F-G. A JC-1 probe was used to stain mitochondria. Then, the red fluorescence and green fluorescence of mitochondria were observed. Nurr1-loaded adenovirus (Ad-Nurr1) and control adenovirus (Ad-Ctrl) were transfected into A549 cells. *p＜0.05.

3.3 Nurr1 overexpression activates mitochondrial apoptosis

Subsequently, we explored whether Nurr1 overexpression activated mitochondrial apoptosis in A549 cells. Mitochondrial apoptosis is characterized by mitochondrial oxidative stress, mitochondrial cyt-c liberation into the nucleus, and caspase-9 activation [46, 48]. First, an immunofluorescence assay was used to observe mitochondrial ROS production. Compared to Ad-Ctrl transfection, Ad-Nurr1 transfection significantly increased mitochondrial ROS production in A549 cells (Fig. 3A-B). To further analyze mitochondrial ROS overloading, we measured alterations in cellular antioxidants. As shown in Fig. 3 C-E, compared to Ad-Ctrl transfection, Ad-Nurr1 transfection reduced the concentrations of cellular antioxidants, including GSH, GOD and GPX. This information illustrated that Nurr1 overexpression triggered mitochondrial oxidative injury in A549 cells.

Figure 3

Nurr1 overexpression triggers mitochondrial apoptosis in A549 lung cancer cells. A-B. Cellular oxidative stress was determined using an immunofluorescence assay with a ROS probe. The fluorescence intensity of ROS was measured. C-E. ELISA was used to analyze the concentrations of cellular antioxidants, such as GSH, GOD and GPX. Nurr1-loaded adenovirus (Ad-Nurr1) and control adenovirus (Ad-Ctrl) were transfected into A549 cells. F-G. Results of an immunofluorescence assay for cyt-c liberation. DAPI was used to stain the nuclei. H-L. Proteins were isolated from A549 cells treated with Ad-Nurr1. Then, western blotting was used to evaluate the expression of mitochondrial apoptosis-related proteins such as caspase-9, Bax, Bcl2 and survivin. *p＜0.05.

In addition to mitochondrial oxidative injury, we further observed alterations in cyt-c translocation into the nucleus (Fig. 3F-G). Through immunofluorescence studies, we found that cyt-c appeared in the cytoplasm in normal A549 cells. Interestingly, Ad-Nurr1 transfection promoted cyt-c liberation into the nucleus, indicating that Nurr1 overexpression evoked cyt-c migration. Subsequently, western blotting was used to confirm the alteration of mitochondrial apoptotic proteins. As shown in Fig. 3H-L, compared to Ad-Ctrl transfection, Ad-Nurr1 transfection upregulated the expression of caspase-9 and Bax. However, the expression of Bcl-2 and survivin were downregulated in response to Nurr1 overexpression. This information indicated that Nurr1 activated mitochondrial apoptosis in A549 cells.

3.4 Nurr1 overexpression promotes mitochondrial fission and inhibits mitochondrial fusion

In addition to mitochondrial dysfunction, we also observed alterations in mitochondrial structure. First, an immunofluorescence assay was used to observe mitochondrial morphology. As shown in Fig. 4A-B, the mitochondria were interconnected in normal A549 cells, and the average mitochondria was ~8.2 μm in length. Interestingly, Ad-Nurr1 transfection induced the formation of mitochondrial debris, and the length of mitochondria was reduced to ~2.9 μm. This information indicated that Nurr1 overexpression disturbed mitochondrial structural homeostasis. Previous studies have reported that mitochondrial structure is closely regulated by mitochondrial fission and mitochondrial fusion. Therefore, western blotting analysis was conducted to evaluate alterations in parameters related to mitochondrial fission and fusion [47, 49]. As shown in Fig. 4C-I, compared to control Nurr1 expression, Nurr1 overexpression upregulated the levels of Drp1, Mff and Fis1, indicating activation of mitochondrial fission in response to Nurr1 overexpression. In contrast, the expression of Mfn1, Mfn2 and Opa1 was downregulated in Nurr1-overexpressing cells, indicating inhibition of mitochondrial fusion in response to Nurr1 overexpression. Altogether, these data indicated that Nurr1 overexpression induced an imbalance between mitochondrial fission and fusion.

Figure 4

Nurr1 modulates mitochondrial fission and mitochondrial fusion in A549 cancer cells. A-B. Immunofluorescence for mitochondria and the mitochondrial structure was observed. Changes in average length of mitochondria were measured in response to Nurr1 overexpression. C-I. Proteins were isolated from A549 cells transfected with Ad-Nurr1, and the expression of mitochondrial fission factors, such as Drp1, Mff and Fis1, was measured. In addition, the levels of mitochondrial fusion factors, including Mfn1, Mfn2 and Opa1, were determined via western blotting in A549 cells transfected with Ad-Nurr1. *p＜0.05.

3.5 Nurr1 activated the p53-Drp1 signaling pathway

Considering the regulatory effects of Nurr1 on mitochondrial homeostasis, we wanted to determine the mechanisms by which Nurr1 modulated mitochondrial homeostasis in A549 cells. Previous studies have identified Drp1 as a master regulator of mitochondrial function, and increased Drp1 expression is associated with cancer cell death and invasion inhibition. In addition, p53 has been found to be the upstream mediator of mitochondrial homeostasis in lung cancer. Based on these data, we wanted to determine whether Nurr1 modulated mitochondrial homeostasis via the p53-Drp1 signaling pathway. First, western blotting was used to observe the influence of Nurr1 on p53 and Drp1 activation. As shown in Fig. 5A-C, compared to Ad-Ctrl transfection, Ad-Nurr1 transfection upregulated the expression of p53; this upregulation was accompanied by an increase in the expression of Drp1. This information illustrated that Nurr1 overexpression could activate the p53-Drp1 signaling pathway. Subsequently, the pathway blocker pifithrin-α (PFTα) was used in Nurr1-overexpressing cells to prevent p53 activation and Drp1 upregulation. The inhibitory effect of PFTα was confirmed via western blotting, as shown in Fig. 5A-C. This result was further supported via immunofluorescence. As shown in Fig. 5D-F, there was little expression of p53 and Drp1 in normal A549 cells. Interestingly, Nurr1 overexpression significantly increased the fluorescence intensity of p53 and Drp1 in A549 cells. In addition, PFTα treatment abolished the promotive effects of Nurr1 on Drp1 activation and p53 upregulation. Altogether, these data indicated that Nurr1 overexpression activated the p53-Drp1 signaling pathway in A549 cells.

Figure 5

Nurr1 overexpression activates the p53-Drp1 signaling pathway. A-C. Western blotting was used to observe the influence of Nurr1 on p53 and Drp1. Nurr1-loaded adenovirus (Ad-Nurr1) and control adenovirus (Ad-Ctrl) were transfected into A549 cells. Pifithrin-α (PFTα), a blocker of p53, was incubated with Nurr1-overexpressing A549 cells. D-F. Results of an immunofluorescence assay for p53 and Drp1. Nurr1-loaded adenovirus (Ad-Nurr1) and control adenovirus (Ad-Ctrl) were transfected into A549 cells. Pifithrin-α (PFTα), a blocker of p53, was incubated with Nurr1-overexpressing A549 cells. *p＜0.05.

3.6 The p53-Drp1 signaling pathway is involved in mitochondrial stress and cancer cell apoptosis

Although we have confirmed the promotive effects of Nurr1 on the p53-Drp1 axis, it is unknown whether the p53-Drp1 signaling pathway is responsible for Nurr1-mediated mitochondrial stress and cancer cell apoptosis. Accordingly, we observed mitochondrial function and cell viability in A549 cells treated with PFTα. As shown in Fig. 6A, compared to control Nurr1 expression, Nurr1 overexpression reduced ATP production, and this effect could be reversed by PFTα, suggesting that Nurr1 reduced ATP production in a manner dependent on Nurr1 activity. In addition, mitochondrial apoptosis, as assessed via immunofluorescence analysis of cyt-c, also demonstrated that Nurr1 promoted cyt-c liberation into the nucleus (Fig. 6B-C). However, PFTα treatment repressed Nurr1-mediated cyt-c translocation. Moreover, Nurr1-mediated LDH release could also be abolished by PFTα treatment (Fig. 6D). This information indicated that Nurr1 induced mitochondrial stress in a manner dependent on the p53-Drp1 signaling pathway. To analyze A549 cell apoptosis, an TUNEL assay was performed. The number of apoptotic cells, as assessed via TUNEL assay, also increased in response to Nurr1 overexpression (Fig. 6E-F). Interestingly, PFTα treatment attenuated Nurr1-mediated A549 cell apoptosis. Taken together, our results indicated that the p53-Drp1 signaling pathway was required for Nurr1-mediated mitochondrial dysfunction and cell death in A549 lung cancer cells.

Figure 6

The p53-Drp1 axis is involved in Nurr1-mediated mitochondrial stress and cell death. A. ATP production was determined in response to Nurr1 overexpression. Pifithrin-α (PFTα), a blocker of p53, was used to prevent the activation of p53 and Drp1 in Nurr1-overexpressing cells. B-C. Results of an immunofluorescence assay for cyt-c in A549 cells. DAPI was used to stain the nuclei. D. Results of an LDH release assay for cell death in A549 cells treated with pifithrin-α (PFTα). E-F. TUNEL staining for apoptotic cells. The number of apoptotic cells was recorded. Nurr1-loaded adenovirus (Ad-Nurr1) and control adenovirus (Ad-Ctrl) were transfected into A549 cells. Pifithrin-α (PFTα), a blocker of p53, was incubated with Nurr1-overexpressing A549 cells. *p＜0.05.