Nuclear factor E2-related factor 2 dependent overexpression of sulfiredoxin and peroxiredoxin III in human lung cancer

Abstract

Background/aims: Oxidative stress results in protein oxidation and is implicated in carcinogenesis. Sulfiredoxin (Srx) is responsible for the enzymatic reversal of inactivated peroxiredoxin (Prx). Nuclear factor E2-related factor 2 (Nrf2) binds to antioxidant responsive elements and upregulates the expression of Srx and Prx during oxidative stress. We aimed to elucidate the biological functions and potential roles of Srx in lung cancer.

Methods: To study the roles of Srx and Prx III in lung cancer, we compared the protein levels of Nrf2, Prxs, thioredoxin, and Srx in 40 surgically resected human lung cancer tissues using immunoblot and immunohistochemical analyses. Transforming growth factor-β(1), tumor necrosis factor-α, and camptothecin treatment were used to examine Prx III inactivation in Mv1Lu mink lung epithelial cells and A549 lung cancer cells.

Results: Prx I and Prx III proteins were markedly overexpressed in lung cancer tissues. A significant increase in the oxidized form of a cysteine sulfhydryl at the catalytic site of Prxs was found in carcinogenic lung tissue compared to normal lung tissue. Densitometric analyses of immunoblot data revealed significant Srx expression, which was higher in squamous cell carcinoma tissue (60%, 12/20) than in adenocarcinoma (20%, 4/20). Also, Nrf2 was present in the nuclear compartment of cancer cells.

Conclusions: Srx and Prx III proteins were markedly overexpressed in human squamous cell carcinoma, suggesting that these proteins may play a protective role against oxidative injury and compensate for the high rate of mitochondrial metabolism in lung cancer.

Keywords: GA-binding protein transcription factor; Lung neoplasms; Peroxiredoxins; Sulfiredoxin.

Figure 1

Expression and cellular localization of peroxir-edoxin (prx) III and sulfiredoxin (srx). A549 cells were stained for Prx III (FITC), Srx (FITC), and mitochondrial fraction (Mitotracker) and then examined by confocal microscopy (A, B). A549 cells were subjected to subcellular fractionation to yield mitochondria-enriched heavy membrane (HM) and cytosolic fractions (CYF). Cell lysates were prepared and subjected to immunoblot analysis with antibodies to either Prx III or Srx (C).

Figure 2

Oxidation of peroxiredoxin (Prx) I, II, and III in Mv-1Lu by transforming growth factor (TGF)-β1. Mv1Lu cells were cultured in DMEM containing 10% FBS and were treated with 2 ng/mL TGF-β1 for the periods indicated. Cell lysates were analyzed with 2D gel electrophoresis, followed by immunoblot analysis with indicated antibodies. Ox, oxidation, re, reduced form.

Figure 3

Sulfiredoxin (Srx) controls peroxiredoxin (Prx) III oxidation in A549 by transforming growth factor (TGF)-β1. Cells were transfected with either a control siRNA or a siRNA specific for human Srx mRNA, after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to Srx or α-tubulin (A). Two days after cells were transfected as in (A), cells were exposed to 10 ng/mL TGF-β1 for 3 days and cell lysates were analyzed with 2D gel electrophoresis, followed by immunoblot analysis with Prx III antibody (B). Ox, oxidation form; Re, reduced form.

Figure 4

Sulfiredoxin (Srx) controls peroxiredoxin (Prx) III oxidation in A549 cells by tumor necrosis factor (TNF)-α and camptothecin. Cells were transfected with either control siRNA or siRNA specific for human Srx mRNA. Two days after transfection, cells were exposed to 10 ng/mL TNF-α for 24 hours (A) or 10 µM camptothecin for 24 hours (B) and cell lysates were analyzed with 2D gel electrophoresis, followed by immunoblot analysis with Prx III antibody. Ox, oxidation form; Re, reduced form.

Figure 5

Increased expression of antioxidant responsive element (ARE)-proteins in human lung cancer tissue (squamous cell carcinoma). Expression of peroxiredoxin (Prx) II, and III as well as thioredoxin (Trx) and sulfiredoxin (Srx) in both lung cancer (C) and paired normal tissue (N) was subjected to immunoblot analysis with antibodies to each, respectively. Expression of nuclear factor E2-related factor 2 (Nrf2) in both lung cancer (C) and paired normal tissue (N) were subjected to subcellular fractionation and the nucleic fraction (NR) was subjected to immunoblot analysis with Nrf2 antibody.

Figure 6

Increased expression of sulfiredoxin (Srx) in human lung cancer tissue: squamous cell carcinoma (A) and adenocarcinoma (B). Expression of Srx in both lung cancer (C) and paired normal tissue (N) were subjected to immunoblot analysis with Srx antibody.

Figure 7

Expression of sulfiredoxin (Srx) in human squamous cell carcinoma. Individual data were quantified as densitometry units (A) and expressed as relative to the corresponding value for expression of Srx in lung cancer and paired normal tissue (B).

Figure 8

Expression of sulfiredoxin (Srx) in human adenocarcinoma. Individual data were quantified as densitometry units (A) and expressed as relative to the corresponding value for expression of Srx in lung cancer and paired normal tissue (B).

Figure 9

Expression of sulfiredoxin (Srx) and survival in all cell types of lung cancer tissue. Data are expressed as relative to the corresponding value for expression of Srx in all lung cancer cell types and paired normal tissue (A). Data are expressed as relative to the corresponding value for expression and survival of Srx in all cell types of lung cancer and paired normal tissue (B).

Figure 10

Expression of sulfiredoxin (Srx) in human lung cancer tissue. Paraffin-fixed 5 M slide of lung cancer tissue was deparaffinized, incubated with Srx antibody, and visualized with DAB as the chromogen (H&E, × 400). Immunohistochemistry of adenocarcinoma (A) and squamous cell carcinoma (B) are shown. black arrow indicates nucleus and white arrow indicates cytoplasm.