Elsevier

Lung Cancer

Volume 39, Issue 1, January 2003, Pages 15-22
Lung Cancer

Expression of antioxidant enzymes in bronchial metaplastic and dysplastic epithelium

https://doi.org/10.1016/S0169-5002(02)00392-6Get rights and content

Abstract

We investigated immunohistochemical expression of manganese superoxide dismutase (MnSOD) and three hydrogen peroxide (H2O2) scavenging pathways, i.e. catalase (CAT), γ-glutamyl cysteine synthetase (γGCS) and thioredoxin (Trx) system in normal bronchial epithelium, bronchial metaplasia and dysplasia and correlated their expression with NF-κB activation (p50) and proliferation (Ki67). Normal bronchial epithelium was positive for MnSOD, heavy and light subunits of γGCS, CAT and Trx and TrxR. Metaplastic epithelium showed strongest expression of γGCSh and Trx, whereas dysplastic epithelium expressed most prominently MnSOD and CAT. There was a significant correlation between expression of γGCSh and γGCSl (P=0.034) and Trx and TrxR (P=0.037). Trx expression also correlated with γGCSh (P<0.001) and γGCSl (P=0.012) and TrxR with γGCSh (P<0.001) but not with γGCSl immunoreactivity (P=0.744). Expression of p50 was highest in metaplastic epithelium while Ki67 was highest in dysplastic lesions. Expression of Trx and γGCSh correlated inversely with age of the patients (R=−0.6038, P<0.001 for Trx and R=−0.6162, P<0.001 for γGCSh). Changes in the expression of these enzymes in bronchial lesions might be due to alterations of antioxidative mechanisms due to irritation via exogenous toxins and activation of reactive oxygen species (ROS) known to be associated with induction of metaplasia and dysplasia in the bronchial tree.

Introduction

Carcinogenesis is a multistep phenomenon with accumulation of genetic changes and an eventual development of invasive cellular clone capable of metastasizing to distant sites. In the bronchial three accumulation of such genetic changes is paralleled by morphological equivalents starting from metaplasic to dysplastic changes of different severity and carcinoma in situ to invasive malignancy with poor prognosis. Genetic changes associated with the development of preneoplastic abnormalities include mutations or dysfunction in the p53 and p16 genes, and bcl-2 [1], [2], [3]. One important factor responsible for the development of the genetic changes in the bronchial epithelium is tobacco smoke. It contains several carcinogens, such as benzopyrene, which is capable in forming adducts to DNA and cause mutations in vital cellular genes. Tobacco smoke also contains reactive oxygen species (ROS), which react with DNA and cause genetic alterations in the cells [4], [5]. Persistent mild oxidant stress also stimulates cell proliferation [6], [7], [8] why it may have important influence on the survival of cells with DNA aberrations.

Given the important role of ROS in causing DNA damage and cell proliferation, antioxidant enzymes (AOEs) and related pathways probably have important influence against these alterations. Such mechanisms include proteins, which bind metals, such as iron and copper, vitamins, such as vitamin E, and a large group of AOEs and related proteins. Superoxide dismutases, such as manganese SOD (MnSOD), CuZnSOD and extracellular SOD (ECSOD) decompose superoxide to hydrogen peroxide (H2O2) [9]. H2O2 can, however, be converted to a potent hydroxyl radical if not decomposed locally. Therefore, cells have an efficient H2O2 scavenging battery with tissue and cell specific distribution and compartmentalization. Most important of these pathways include peroxisomal catalase (CAT), and cytosolic and extracellular glutathione peroxidases (GPX), which latter enzymes require glutathione (GSH) for H2O2 removal. [9]. Other interesting enzymes involved in the decomposition of H2O2 and maintenance of the cellular redox-state include the thioredoxin (Trx)–thioredoxin reductase (TrxR) system and small cysteine containing proteins thioredoxin peroxidases (peroxiredoxins) which have important associations also with cancer biology [9], [10], [11].

Manganese superoxide dismutase (MnSOD) is one of the most important AOEs in living cells. It is synthesized in the cytoplasm as a precursor molecule containing a leader signal which is removed during the transport of the molecule to the mitochondria. It is highly inducible by cytokines and oxidants [12], [13], and it has been detected in human bronchial epithelium and alveolar pneumocytes [14]. Several in vitro studies and experimental models have shown that MnSOD can reduce tumor growth and proliferation [15]. It has also been suggested that MnSOD may be tumor suppressor gene On the other hand, there are also investigations showing increased expression of MnSOD in several tumors [16], [17], [18]. MnSOD overexpression can also suppress apoptosis at least in cultured cells in vitro [19].

Glutathione (l-γ-glutamyl-l-cysteinyl-glycine) (GSH) is one of the most important antioxidants in human lung [20]. For instance its concentration in the epithelial lining fluid is remarkably higher than in plasma. Experimental models have also shown GSH to be critical antioxidant in protecting airway epithelium from oxidant injury [21]. The rate-limiting enzyme in GSH synthesis is γ-glutamyl cysteine synthetase (γGCS). γGCS is a cytosolic protein consisting of heavy (γ-GCSh 73 kDa) and light (γ-GCSl 30 kDa) chains [20], [21]. The heavy chain is also called as catalytic subunit since it contains the catalytic activity of the enzyme and light chain as regulatory subunit [21], [22]. Both subunits are induced by acute exposures to oxidants, cigarette smoke, and oxidative stress associated inflammatory mediators such as tumor necrosis factor alpha [20], [23]. The kinetics of γGCS suggests its importance especially at relatively low, physiological H2O2 concentrations.

CAT is located mainly in peroxisomes but it has been detected also in the cytosolic fraction of neutrophils. It is mainly constitutive, i.e. it is not induced by oxidants or cytokines as is the case with MnSOD and γGCS. It has higher Km for H2O2 than GPX suggesting that it may have importance especially at high H2O2 concentrations [9]. In human lung, CAT has been detected in bronchial epithelium, alveolar epithelium especially in pneumocyte II cells and in alveolar macrophages [24], [25], and it appears to be responsible for the majority of H2O2 consumption of alveolar macrophages in vitro [26].

Trx group of proteins is cysteine containing protein family with efficient antioxidative, anti-apoptotic and proliferative capacity [10]. Trx is mainly cytosolic, but can be translocated to nucleus by binding to NF-κB. In addition to the cytosolic Trx (Trx1), another Trx (Trx2) has been described which is localized to mitochondria, and additional Trx-like cytosolic protein (p32TrxL) has been recently characterized [27]. Trx increases DNA binding of NF-κB [28] the expression of glucocorticoid receptor-mediated genes and gluocorticoid receptor [29], and AP-1 activity [30]. Trx is activated by a variety of stress stimuli such as hypoxia, lipopolysaccharide, H2O2, infections, photochemical and UV irradiation [27], [28]. Active Trx system requires thioredoxin reductase activity (TrxR), which is flavin adenine dinucleotide containing protein with C-terminal selenocysteine residue. Two TrxRs have been characterized to date [27], [28]. Trx and TrxR have been detected in human bronchial epithelium [28], [31].

Given the inducibility of most these enzymes by oxidants and cytokines, it can be hypothesized that these enzymes may be overexpressed in various inflammatory states of human lung. Induction of these enzymes not only protects cells against oxidants but may also give advantage for cell proliferation during persistent oxidant exposure. On the other hand, CAT, which is not induced by cytokines, can be hypothesized to be expressed similarly both in the diseased and healthy tissue.

In this study we assessed whether these enzymes are also induced in the preneoplastic lesions of human lung; MnSOD, γGCSh and γGCSl, CAT, Trx and TrxR were investigated in healthy bronchial epithelium and in the metaplastic and dysplastic lesion of bronchial epithelium. The results were also correlated with proliferation, as detected by immunohistochemistry of Ki67, and the expression of p50 as an indicator of NF-kB activation.

Section snippets

Tissue material

Thirty-four cases of bronchial dysplasia (n=17) and metaplasia (n=17) were collected from the files of Department of Pathology, Oulu University Hospital. Of dysplasias, seven were mild, seven moderate and three severe. The criteria for the diagnosis of dysplasia were according to WHO [32]. The average age of the patients was 62.1±10.4 years. In the dysplasia material, there were 16 males and one female. Similarly in the metaplasia material, there were 13 males and four females. Additionally

MnSOD

MnSOD expression was seen in the cytoplasm of the bronchial cells. 6.2% of normal bronchial cells and 11.1% of metaplastic cells expressed positivity. In dysplasia the percentage was 13.1% and there was a statistically significant difference between dysplasia and normal epithelium (P=0.049) (Fig. 1A). The expression of MnSOD in the bronchial epithelium of non-smokers was somewhat higher (8.9%) but was not statistically significantly different from other cases of normal epithelium (P=0.54).

γ-Glutamylcysteine synthetase

The

Discussion

We investigated the expression of MnSOD, γGCSh and γGCSl, CAT, and Trx and TrxR in a set of bronchial dysplasias and metaplasias and samples from normal bronchial epithelium. The results show that expression of these enzymes is generally somewhat elevated in metaplastic and dysplastic epithelium of the bronchial three. A clear rising tendency in immunoreactivity was found for MnSOD and there was a statistically significant difference between the expression of dysplastic and normal bronchial

Acknowledgements

The authors thank Päivi Koukkula and Manu Tuovinen for their excellent technical assistance. We also thank Professor A. Holmgren for providing the anti-TrxR antibody and Professor J.D. Crapo for antibodies to MnSOD and Dr Kavanagh for providing the anti γ-GCS antibodies. This work was financially supported by the University of Oulu, Finnish Anti-Tuberculosis Association Foundation, Juselius Foundation and the Cancer Society of Finland.

References (37)

  • K. Nuorva et al.

    Concurrent p53 expression in bronchial dysplasias and squamous cell lung carcinomas

    Am. J. Pathol.

    (1993)
  • C. Walker et al.

    Expression of the BCL-2 protein in normal and dysplastic bronchial epithelium and in lung carcinomas

    Br. J. Cancer

    (1995)
  • N. Banzet et al.

    Tobacco smoke induces mitochondrial depolarization along with cell death: effects of antioxidants

    Redox. Rep.

    (1999)
  • S. Loft et al.

    Cancer risk and oxidative DNA damage in man

    J. Mol. Med.

    (1996)
  • B. Halliwell et al.

    Free radicals in biology and medicine

    (1989)
  • S.G. Rhee

    Redox signaling: hydrogen peroxide as intracellular messenger

    Exp. Mol. Med.

    (1999)
  • V.L. Kinnula et al.

    Generation and disposal of reactive oxygen metabolites in the lung

    Lab. Invest.

    (1995)
  • G.H. Wong et al.

    Induction of manganous superoxide dismutase by tumor necrosis factor: possible protective mechanism

    Science

    (1988)
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