PRDX5 as a novel binding partner in Nrf2-mediated NSCLC progression under oxidative stress

PRDX5 interacts with Nrf2 in NSCLC tissues and cell lines

We first analyzed the expression of PRDX5 in both mRNA and protein levels in five lung cancer cell lines (A549, H460, NCI-H1299, Calu1 and SK-MES-1) by using qRT-PCR and Western blot method, respectively, and found that the expression of PRDX5 mRNA and protein were all higher than that in the normal bronchial epithelial cell 16-HBE, which increased the likelihood that PRDX5 may participated in the malignant progression of NCSLC (Figure 1A and 1B). Then, by immunoprecipitations, we found PRDX5 interacts with Nrf2 in both NSCLC tissues and cell lines. Figure 1C showed that PRDX5 can be brought down by an antibody of anti-Nrf2 from fresh NCSLC tissue lysate, and vice versa. At the same time, in PRDX5-expressing non-tumorous lung tissues, we also found that Nrf2 could be immune-precipitated by PRDX5, indicating that the binding of the two proteins was not limited to NCSLC tissues. When we treated A549 and H1299 cell lines with H₂O₂, we detected that there was obvious interaction between Nrf2 and PRDX5 (Figure 1D). Similarly, we also observed that the two proteins could bind to each other in untreated NCSLC cells. (Figure 1D). To further confirm Nrf2 and PRDX5 colocalization, immunofluorescence analysis was conducted in A549 and H1299 cells. The results showed Nrf2 and PRDX5 were partially co-localized in H₂O₂-treated NCSLC cells (Figure 1E). All these findings indicated that Nrf2 interacted with PRDX5 in NCSLC tissues and NCSLC cells.

Figure 1. PRDX5 interacted with Nrf2 in NSCLC tissues and related cell lines. (A) qRT-PCR analysis of PRDX5 mRNA level in NSCLC cell lines and 16-HBE cells. The data are reported as the mean ± SD. *P < 0.05, compared with the level in 16-HBE cells. (B) PRDX5 proteins in the different NSCLC cell lines and the normal bronchial epithelial cell 16-HBE analyzed by Western blot analysis. The data shown represent the mean ± SD (*P < 0.05, compared with the level in 16-HBE cells). (C) Reciprocal immunoprecipitation of Nrf2 and PRDX5 in human NSCLC tissue (figure above) and PRDX5 was immunoprecipitated using an anti-Nrf2 antibody in the adjacent normal tissue (figure below). Lysates of the tissues were immunoprecipitated with anti-Nrf2, anti-PRDX5 antibodies or control IgG. The immunoprecipitates were subjected to Western blot analysis with anti-PRDX5 and anti-Nrf2 antibodies. (D) Interaction between Nrf2 and PRDX5 in A549 and NCI-H1299 cells under H₂O₂ treatment or nontreatment. The lysates obtained from the cells treated with 100 μM H₂O₂ for 30 min or not were immunoprecipitated using anti-Nrf2, anti-PRDX5 antibodies or control IgG. (E) Immunofluorescence analysis of Nrf2 and PRDX5 in A549 and NCI-H1299 cells. A549 and H1299 cells were pre-incubated with 100 μM H₂O₂ for 30 min, and then immunostained with a combination of anti-Nrf2 and anti-PRDX5 antibodies. The fluorescent images were digitally merged. Yellow coloration in overlay panels indicates colocalization of Nrf2 and PRDX5. Nuclei were counterstained with DAPI. Scale bar, 50 μm.

Nrf2-mediated recruitment of PRDX5 enhanced NQO1 expression

We treated A549 and H1299 cells with H₂O₂ and found the expression level of NQO1 protein increased significantly, while knockdown of Nrf2 reverse the upregulation of NQO1 protein in this circumstance of stimulation with H₂O₂ (Figure 2A). The above results showed that Nrf2 mediated the effect of H₂O₂. Similarly, PRDX5 knockdown significantly reduced NQO1 protein expression level in H₂O₂ treated A549 and H1299 cells (Figure 2A). Further, we try to use cycloheximide (CHX) chase experiment to clarify the mechanism underlying Nrf2/PRDX5-induced augmented NQO1 protein expression. The results showed that when treated with H₂O₂ or not in A549 and H1299 cells, the half-life time of NQO1 protein performed equally, and the results indicated that enhanced NQO1 protein expression stimulated with H₂O₂ did not occur at its post-translational level (Figure 2B). In sum, we clarified that Nrf2-mediated recruitment of PRDX5 enhanced NQO1 expression in NCSLC cells from the above results.

Figure 2. The influence of Nrf2/PRDX5 on NQO1 expression. (A) A549 and NCI-H1299 cells transfected into Nrf2 shRNA or PRDX5 shRNA were treated with serum-free medium overnight. The serum-starved cells were mock-treated, or stimulated with 100 μM H₂O₂ for 12 h. The expressions of Nrf2, PRDX5 and NQO1 were determined by Western blot. The data are mean ± SD (*P < 0.05). (B) After stimulated with 100 μM H₂O₂ for 12 h, A549 and H1299 cells were treated with 25 mg/L of cycloheximide (CHX) for the indicated period of time and subjected to Western blot analysis.

Depletion of PRDX5 suppresses Nrf2-mediated cell proliferation

We first tested and verified the impact of Nrf2 shRNA on the proliferation of NCSLC cells. The results of CCK-8 assay showed that the group treated with H₂O₂ elicited a significant increase in the proliferation of A549 and H1299 cells, while knockdown of Nrf2 with shRNA suppressed the proliferating effect obviously (Figure 3A). Colony formation assay also indicated the same effect of Nrf2 shRNA on cell proliferation in vitro (Figure 3B). Then we analyzed PRDX5 and NQO1 expression pattern in different proliferating statuses of NCSLC cells. The results showed that the protein levels of PRDX5 and NQO1 were increased gradually in released both A549 and H1299 cells after 72h of serum starvation (Figure 3C). These data support the conception that PRDX5 and NQO1 played important roles in regulating NCSLC proliferation. Next, we investigated whether the role of Nrf2 in promoting NCSLC growth is related to PRDX5 and NQO1. As shown in Figure 3D and 3E, the results illustrated that knockdown of PRDX5 or NQO1 significantly attenuated proliferation and colony formation capacity induced by H₂O₂ in A549 and H1299 cells, suggesting that Nrf2 may exert the effect of promoting proliferation through regulating PRDX5-dependent NQO1 expression. Then, we also analyzed the effects of Nrf2 and/or PRDX5 shRNA on cell proliferation and apoptosis of A549 and H1299 under oxidative stress by using flow cytometry. The results showed that Nrf2 and/or PRDX5 shRNA significantly increased apoptosis ratio of A549 and H1299 cells treated with H₂O₂ while decreased cell proliferation (Figure 4A, 4B). Subsequently, we further employed the EdU incorporation assay to determine the effects of Nrf2 and/or PRDX5 shRNA on cell proliferation. The results also showed that Nrf2 and/or PRDX5 shRNA groups significantly decreased cell proliferation ratio of A549 and NCI-H1299cells treated with H₂O₂ (Figure 5A, 5B).

Figure 3. Nrf2 enhanced the growth of NSCLC cells by PRDX5 and NQO1. (A) Growth curves of A549 and H1299 cells treatment with 100 μM H₂O₂ or knockdown of Nrf2 using CCK-8 assay. All the data are mean ± SD and representative of three independent experiments (*P < 0.05). (B) Effect of PRDX5 or NQO1 knockdown on proliferation of A549 and H1299 cells in the presence of 100 μM H₂O₂ analyzed by CCK-8 assay. The data are reported as the mean ± SD of three independent experiments (*P < 0.05). (C) Nrf2 had an effect on the colony formation ability of NSCLC cells. Equal numbers of A549 and H1299 cells after treatment as above were seeded onto 6-well plates. The cells were fixed and stained with crystal violet after 14 days. The cell colonies (>0.5 mm in diameter) were counted after staining (mean ± SD, *P < 0.05). (D) Colony-forming capability was measured by colony formation assay in A549 and H1299 cells after transfected with PRDX5 shRNA or NQO1 shRNA and stimulated with 100 μM H₂O₂. The bar chart showed the number of colonies (>0.5 mm in diameter) in A549 and H1299 cells (mean ± SD, *P < 0.05). (E and F) A549 and H1299 cells were serum starved for 72 h, and then refed with serum for 0, 8, 16 and 24 h. The cell lysates of the corresponding time point were prepared and analyzed by Western blot using antibodies against PRDX5 and NQO1. GAPDH was used as a loading control. The bar charts demonstrated the ratio of PRDX5 or NQO1 to GAPDH by densitometry in A549 and H1299 cells. Mean ± SD of three independent experiments.

Figure 4. The effects of Nrf2 and/or PRDX5 shRNA on cell proliferation by EdU incorporation assay. (A) Nrf2 and/or PRDX5 shRNA significantly decreased cell proliferation ratio of A549 cells treated with H₂O₂ (*P < 0.05). (B) Nrf2 and/or PRDX5 shRNA significantly decreased cell proliferation of NCI-H1299 cells treated with H₂O₂ (*P < 0.05).

Figure 5. The effects of Nrf2 and/or PRDX5 shRNA on cell proliferation and apoptosis of A549 and H1299 under oxidative stress by using flow cytometry. (A) Nrf2 and/or PRDX5 shRNA significantly increased apoptosis ratio of A549 and H1299 cells treated with H₂O₂ (*P < 0.05). (B) Nrf2 and/or PRDX5 shRNA significantly decreased cell proliferation of A549 and H1299 cells treated with H₂O₂ (*P < 0.05).

Clinical correlation of PRDX5 and Nrf2 expression in NCSLC tissues

To study clinical correlation, we first analyzed Nrf2, PRDX5 and NQO1 mRNA expression in both tumor tissues and the adjacent normal tissues, and the results showed upregulated these mRNAs in tumors (Figure 6A). The statistical correlations analysis among Nrf2, PRDX5 and NQO1 expressions in NSCLC samples showed that the level of PRDX5 expression in NCSLC tissues correlated with Nrf2 and NQO1 expression level very weakly (Figure 6B), while by subgroup of NSCLC patients with clinical TNM stage III-IV comparison, we found strong correlation among the three mRNA levels (Figure 6C). Further, we collected 26 pairs of matched fresh NCSLC and adjacent normal tissues to analyze PRDX5 and Nrf2 protein levels by using western blot. The results showed that the expression of PRDX5 increased significantly in cancer samples than that of in the non-tumorous samples, and the same trend of Nrf2 and NQO1 protein levels compared to the adjacent normal ones (Figure 7A). Moreover, we analyzed by immunohistochemistry analysis in 121 NCSLC specimens to further explore the clinical relevance between expression of Nrf2/PRDX5 and the clinicopathological factors (Figure 7B). The next study showed that PRDX5 and Nrf2 expressions were tightly associated with tumor size, clinical TNM stage, lymph node infiltration, differentiation and Ki-67 expression (Table 1).

Figure 6. The correlation of Nrf2, PRDX5 and NQO1 mRNA expression in NSCLC specimens. (A) Nrf2, PRDX5 and NQO1 mRNA expressions in both tumor tissues and the adjacent normal tissues (**P < 0.01, compared with the adjacent normal tissues). (B) Statistical correlations among Nrf2, PRDX5 and NQO1 expressions in NSCLC samples. (C) Statistical correlations among Nrf2, PRDX5 and NQO1 expressions in NSCLC patients with Clinical TNM stage III-IV.

Figure 7. The protein expressions of Nrf2, PRDX5 and NQO1 in NSCLC specimens. (A) The Nrf2, PRDX5 and NQO1 expressions in 26 NSCLC and the adjacent normal tissues by western blot analysis. The representative western blot results in 6 cases are shown. GAPDH was used as a control for protein load and integrity. The bar chart demonstrated the ratio of Nrf2, PRDX5 and NQO1 expression to GAPDH between tumor and non-tumor tissues for the above by densitometry. *,^#P < 0.05, significant upregulated expression of Nrf2, PRDX5 and NQO1 in cancerous tissues, compared with adjacent normal tissues. The data are reported as the mean ± SD (*, ^#P < 0.05, compared with the adjacent tumor tissues). (B) Immunohistochemical analysis of Nrf2, PRDX5 and NQO1 in NSCLC tissues (a–d) and the adjacent normal tissues (e–g). Scale bar, 100 μm. (C) Cumulative survival curves according to Nrf2 and PRDX5 expression in 121 patients with NSCLC. Left, overall survival curves of low PRDX5 expression group vs high PRDX5 expression group (the choosing relative level of 2.323 as the optimal cut-off point of PRDX5). Middle, overall survival curves of low Nrf2 expression group vs high Nrf2 expression group (the choosing relative level of 2.825 as the optimal cut-off point of Nrf2). Right, overall survival curves of high Nrf2/PRDX5 expression group vs the other group.

Finally, Kaplan–Meier analysis showed that higher expressions of Nrf2 or PRDX5 were prone to obtain lower overall survival rates in NCSLC patients (P = 0.002 and 0.006), and high expressions of both Nrf2 and PRDX5 suggested poor prognosis of patients with NCSLC (P = 0.001, Figure 7C).

Therapeutic effects of Nrf2 and PRDX5 shRNA on tumor growth in vivo

We established human lung adenocarcinoma A549 xenografts mice models. In short, suspended A549 cells (5 × 10⁶) in 0.2 mL volume of Matrigel (BD Biosciences, San Jose, CA), then injected them into two flanks of nude mice subcutaneously. The nude mice were anesthetized when tumor volume gained to about 100 mm³, treated with Nrf2 and/or PRDX5 shRNA. After treatment period of 25 days cycle, the tumor volumes of Nrf2 and PRDX5 shRNA group were significantly decreased than that of the other groups and weights of the corresponding tumor were also the smallest (^*P < 0.05, ^**P < 0.01, Figure 8A–8C). Then we detected the protein expressions of Nrf2, PRDX5 and NQO1 in xenograft tumors in each group, and found the expression level of each protein in the treatment group was significantly lower than that in the corresponding control group (Figure 8D, 8E). The results indicated that Nrf2 and PRDX5 may be potential effective therapeutic targets.

Figure 8. Effect of Nrf2 and PRDX5 shRNA on tumor growth in vivo. (A) Representative xenografts in each group. (B) The weights of xenograft tumors in mice after treatment with each shRNA. (*^,#P < 0.05, **P < 0.01). (C) Antitumor therapeutic efficacy in tumor bearing mice (*P < 0.05, **P < 0.01). (D and E) The protein expressions of Nrf2, PRDX5 and NQO1 in xenograft tumors in each group. (*^,#,&P < 0.05, compared with the control group, respectively).

Patients and tissue specimens

NSCLC tissues and the adjacent normal tissues were collected from 121 patients who underwent lung cancer resection at Affiliated Hospital of Nantong University between 2009 and 2012. The Department of Pathology of Affiliated Hospital of Nantong University evaluated the tissue samples in accordance with World Health Organization standards. In addition, the clinical and follow-up data from patients’ medical records were collected. 26 pairs of fresh NSCLC and adjacent normal tissue samples were frozen in the liquid nitrogen immediately after surgical removal until using for Western blot analysis. The Ethics Committee of Affiliated Hospital of Nantong University (Jiangsu, China) approved the present study in accordance with the Declaration of Helsinki. Tissue samples from patients enrolled in the study were collected after obtaining informed consent signed by the patients or their family members.

Cell lines

The human lung cancer cell lines A549, H460, NCI-H1299, Calu1, SK-MES-1 and the normal bronchial epithelial cell 16-HBE were purchased from the Shanghai Cell Bank of the Chinese Academy of Science (Shanghai, China). Cells were cultured in DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), and incubated in a humidified atmosphere at 37°C and in a humidified atmosphere of 5% CO₂.

Nrf2 and PRDX5 shRNA

Nrf2 short shRNA lentiviral particles (sc-37030-V) and PRDX5 lentiviral particles (sc-40837-V) were purchased from Santa Cruz Biotechnology. The shRNA lentivirus was added to cultured cells for 24h, following by puromycin (5.0 mg/mL)-mediated selection for 10 days. In the stable cells over 95% Nrf2, PRDX5 or NQO1 knockdown was confirmed by Western blotting.

Detection by qRT-PCR

The mRNA levels of PRDX5, Nrf2, NQO1 and GAPDH were analyzed by qRT-PCR using the SYBR Green Supermix kit (TaKaRa Bio, Shiga, Japan), GAPDH as internal reference, and the comparative cycle threshold (Ct) method was used to calculate relative amount of target mRNAs. Sequences of the primer for the genes are shown in Table 2.

Immunofluorescence assay

Immunofluorescence assay according the following steps: first the cells fixed in 4% paraformaldehyde for 0.5 hour at room temperature, PBS buffer washing for three times, and blocked with 5% donkey serum for 2 hours at room temperature, then incubated with anti-Nrf2 and anti-PRDX5 antibodies (Abcam) at 4°C overnight. PBS buffer washing for three times, the cells then were incubated with Alexa Fluor 488 or 594 conjugated secondary antibodies (Abcam) for 1 hour at room temperature. DAPI (Sigma-Aldrich) was used for nuclear staining. After adding anti-fluorescence quenching agent seal, the cells were visualized by the fluorescence microscopy (Leica DMR 3000; Leica Microsystem, Bensheim, Germany).

Western blotting

The harvested cells or tissues were lysed in RIPA buffer containing protease inhibitors (Promega, Madison, WI). The concentration of total protein was determined by BCA protein assay (Bio-Rad, Hercules, CA). Then protein samples containing 30 μg of total protein were separated by SDS-PAGE gels and transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% skimmed milk at room temperature for 1 hour and incubated overnight with the primary antibodies diluted 1:1000 at 4°C. Primary antibodies include PRDX5, Nrf2, NQO1, Flag, and GAPDH (all Abcam, USA). The next day, the membranes were further incubated with secondary antibodies (Rockland Gilbertsville, CA). The blots were scanned by Odyssey (Li-COR, USA) and taken the gray scale value for statistics.

Coimmunoprecipitation

For coimmunoprecipitation, the supernatants of NSCLC tissue or cell lysates were incubated with the primary antibodies or control IgG in conjunction with Protein G Sepharose. Then the collected precipitates were separated and analyzed by Western blot.

Immunohistochemistry

In brief, fix, embed, and cut tissue samples into sections about 4 μm thick. Then deparaffinize, hydrate, repair, and incubate them overnight at 4°C with anti-PRDX5, anti-Nrf2 and anti-NQO1 antibodies diluted in 1:50. Mayer's haematoxylin was used for nuclear counter staining. Two pathologists examined the immunostaining results independently, and the standard stage of tissue samples were divided as follows: 0 of no staining, 1 of weak, light yellow, 2 of moderate, yellow brown, and 3 of strong, brown. High expression as an intensity score of 2, and low expression as a score less than 2.

Cell proliferation assay

For CCK8 detection, cells were cultured in 96-well plates (2 ×10³ cells per well) for 24 hours. The cell number was determined using the cell counting kit 8 (KeyGen, Nanjing, China) at 24, 48, 72, and 96 hours after shRNA transfection.

For 5-Ethynyl-2′-deoxyuridine (EdU) incorporation assay, in brief, cells of 4 × 10³ per well were cultured in 96-well plates for 48 h at 37 °C followed by 50 μM of EDU medium of 100 μL being added per well for another 2 hours at 37 °C, and then be fixed with 4% paraformaldehyde. The fixed cells were permeabilized with 0.5% Triton X-100 for 10 min followed by staining with Apollo® 567 and Hoechst33342, respectively.

Clone formation assay

For plate clone formation assay, cells were plated in 6-well plates at a density of 1000 cells/dish with 5% CO₂ at 37°C for two weeks. Then be washed with PBS, fixed for 15 min with paraformaldehyde and stained with 0.5% crystal violet. Finally, counted and analyzed the number of stained clones.

Flow cytometry assay

The cells were trypsinized and harvested. Apoptosis was stained by using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis kit (Invitrogen, Carlsbad, CA) and cell cycle was stained by a PI-based cell cycle kit (Invitrogen, Carlsbad, CA). Cells were assessed by Attune Acoustic Focusing Cytometer (Invitrogen).

Animal study

The growth inhibited effect by PRDX5 and Nrf2 silencing on NCSLC cell line was determined in vivo. Nude mice were bought from the Experimental Animal Centre of Nantong University. The A549 cell was cultured, collected and washed with PBS buffer, then injected into the flanks of the mice subcutaneously (5×10⁶ cells/200 μL per side, 6 per group). The nude mice were anesthetized when tumor volume gained to about 100 mm³, injected with Nrf2 and/or PRDX5 shRNA. The tumor volumes were measured and calculated every 5 days using the following formula: volume V = width² ×length/2. The experiments were approved by Nantong University Animal Care and Utilization Committee.

Statistical analysis

Statistical analysis was performed by using SPSS 17.0 software package. All values were expressed as the mean ± standard deviation (SD). Differences were analyzed with one-way analysis of variance (ANOVA). When ANOVA detects significant differences, the data of above variables of each experimental group were compared with that of the control group using by a Dunnett’s t-test as post hoc test. The association between Nrf2 and PRDX5 and the clinicopathological factors was analyzed by using the Chi-square (χ²) test. Each experiment was repeated at least three times and differences were considered statistically significant at P < 0.05.