Nrf2 promotes progression of non-small cell lung cancer through activating autophagy

Abstract

The transcription factor, NFE2-related factor 2 (Nrf2) and autophagy have been implicated in the oxidative-stress response during tumor evolution. However, few studies focus on crosstalk between Nrf2 and autophagy in cancer progression of non-small cell lung cancer (NSCLC). Herein, we evaluated the effect of Nrf2 on autophagy in NSCLC and their role in development of NSCLC. Effect of Nrf2 on overal survival (OS) of NSCLC patients were evaluated. Cell biological behaviors in response to Nrf2 were evaluated by MTT, colony formation assay and flow cytometry. Effect of 3-MA (a classical inhibitor of autophagy) on 95D-Nrf2 cells was also analyzed using flow cytometry. After up/down-regulating Nrf2 in NSCLC cell lines, expression of autophagy-related proteins were evaluated with western blot analysis. The results revealed that Nrf2 was an independent prognositc factor negtively associated with OS of NSCLC patients. Elevated Nrf2 expression promotes NSCLC progression, enhancing the escape of tumor cells from apoptosis in vivo and in vitro. Double staining with Annexin V-APC and 7-AAD showed that the proportions of apoptotic cells in 95D-Nrf2 cells were gradually increased after the addition of 3-MA. Importently, Nrf2 induced autophagosome formation and enhanced autophagic activity, which subsequently inhibits NSCLC cell apoptosis. In conclusion, our present study demonstrates that Nrf2 promotes progression of non-small cell lung cancer through activating autophagy. It provides novel insights into Nrf2-mediated of cell proliferation in NSCLC and may facilitate therapeutic development against NSCLC.

Keywords: Nrf2; apoptosis; autophagy; cell proliferation; lung cancer.

Figure 1.

Effect of Nrf2 staus, TNM, and tumor size on OS of NSCLC patients. Median survival time (MS) of NSCLC patients with Nrf2 (−) and Nrf2 (+) was 56 months (95% CI, 50.1 to 61.9) and 17 (95% CI, 14.76 to 19.25), respectively, P = 0.01. The MS of patients with TNM I, II and TNM III, IV was 56 months (95% CI, 50.73 to 61.27) and 17 (95% CI, 14.64 to 19.36), respectively, P = 0.00. The MS of patients with advanced TNM tumor size < 5cm and ≥ 5 cm was 43 months (95% CI, 31.43 to 54.57), and 17 (95% CI, 8.38 to 13.62), respectively, P = 0.00.

Figure 2.

Immunohistochemical staining of Nrf2 protein in NSCLC tissues (magnifcation, x200). Immunohistochemical expression of Nrf2 in paired normal lung tissues (A1-A2), lung adenocarcinoma (C1-C4) and lung squamous cell carcinoma (B1-B4). (B2,C2) weak expression, (B3,C3) moderate expression and (B4,C4) strong expression. Nrf2, NFE2-related factor 2; NSCLC, non-small cell lung cancer.

Figure 3.

The expression of Nrf2 in human NSCLC cell lines. (A) The expression of Nrf2 detected by Western blot in different cell lines; (B) Western blot analysis for Nrf2 protein expression in Nrf2 pcDNA-affected 95D and shRNA-transfected A549 NSCLC cells; (C) Quantifcation of the relative expression(Nrf2/β-actin) of Nrf2 in NSCLC cells. (D)Quantifcation of the relative expression(Nrf2/β-actin) of Nrf2 protein in Nrf2 pcDNA-affected 95D and shRNA-transfected A549 NSCLC cells. The data are presented as mean ± SD of 3 independent experiments.The bar graph shows the relative protein expression (Nrf2/β-Actin in each cell line, *, P < 0.05, **, P < 0.01 compared with the control cells).

Figure 4.

Effects of Nrf2 expression on the proliferation of NSCLC cells in vitro. (A) MTT assay; (B) Colony formation assay. Colonies were counted 14 d later and the number of cells in a colony is more than 50; (C) Cell cycle distribution was analyzed by flow cytometry; (D) Apoptotic and necrotic cells were counted by flow cytometry. Data are presented as mean ± SD of 3 independent experiments. (*, P < 0.05; **, P < 0.01 and ***, P < 0.001 VS.the corressponding control).

Figure 5.

Activities of Nrf2 in NSCLC cells in tumor xenograft models. (A) Photomicrograph of tumors in the different treatment groups; (B) Tumor growth curve in different groups; (C) Immunohistochemical analysis of Nrf2 and autophagy related genes in tumor xenografts. Nrf2 expression in xenografts resulted in the upregulation of beclin1 and LC3 expression (× 200 magnification). Data are presented as mean ± SD of 3 independent experiments. (*, P < 0.05, **, P < 0.01).

Figure 6.

Nrf2 promotes autophagy in NSCLC cells. (A) Endogenous ROS levels in NSCLC cell lines with DCF-DA probe. The intensity fluorescence were counted by flow cytumetry, and the mean intensity of fluorescence in the cells with different groups represents endogenous ROS levels in NSCLC cell lines; (B) Western blot analysis of autophagy-related genes; C. Effects of Nrf2 expression on the regulation of the number of autophagosome with TEM. Data are presented as mean ± SD of 3 independent experiments.

Figure 7.

Effect of ROS on expression of Nrf2. (A) Flow cytometry verified that the level of intracellular ROS in cancer cells gradually increased along with high concentration of H2O2. (B) After treatment A549 cells with H2O2 for 24h. The results showed that the expression of Nrf2 increased after treating with ROS in dose dependent manner.

Figure 8.

Nrf2 promotes NSCLC cell proliferation by activing autophagy. Western blotting for LC3I/II and Beclin1 in 95D-Nrf2 cells treated with 3-MA (0,2.5,5,10 mmol/L); B. Effects of inhibited autophagy on the regulation of the proportions of apoptotic cells in 95D-Nrf2 cells. Apoptosis 005,control (95D-Nrf2 cells untreated with 3-MA); Apoptosis 006,007,008, 95D-Nrf2/3-MA(95D-Nrf2 cells treated with 2.5,5,10 mmol/L 3-MA,respectively). Data are presented as mean ± SD of 3 independent experiments. *, P < 0.05 compared with the control cells.