Reduced expression of endothelial and inducible nitric oxide synthase in a human breast cancer cell line which has acquired estrogen independence
Introduction
The exact role of nitric oxide (NO) in tumor biology has not yet been fully elucidated. This is due to the fact that NO has been implicated in many different aspects of cancer biology including both pro- and anti- tumor functions.
NO may have a role in carcinogenesis by inducing DNA strand breaks [1] and by impairing the tumor suppressor function of p53 [2]. It has also been implicated as part of a signalling cascade for neovascularization [3] and it can increase tumor blood flow [4]. The presence of inducible NOS (iNOS) has been correlated with metastatic disease [5] and a NOS inhibitor significantly reduces bone metastasis [6].
With regard to its anti-tumor role, NO has a cytostatic/cytotoxic role towards tumor cells [7], [8]. Cytotoxic effects are mediated via interference with DNA replication and several enzymes, including aconitase and ubiquinone oxireductase [9]. NO has also been shown to induce apoptosis in tumor cells [10], [11].
The balance between these opposing roles may depend upon the local concentration of NO, with high concentrations of NO exerting anti-proliferative effects and low concentrations facilitating tumor growth [3].
Although tamoxifen is still the treatment of choice for breast cancer patients with estrogen receptor (ER)-positive tumors [12] nearly all patients will eventually become resistant to this treatment [13]. Treatment failure may be associated with a variety of changes in tumor characteristics leading to a more malignant phenotype, including the development of anti-estrogen resistance and progression to estrogen independence.
We have previously characterized two such variants of the ZR-75-1 human breast cancer cell line. In ZR-75-9a1 cells [14] which have acquired tamoxifen resistance, there is loss of detectable ERs and progesterone receptors (PGR), and an increase in epidermal growth factor receptor (EGFR) expression [15]. The ZR-PR-LT cell line [16], which is an estrogen-independent variant, expresses high numbers of PGR and has reduced EGFR expression.
We have recently reported the presence of iNOS in the ZR-75-1 human breast cancer cell line [17] and the aim of the current study was to investigate expression of both endothelial NOS (eNOS) and iNOS, in two more malignant variant cell lines. We report that the tamoxifen resistant ZR-PR-9a1 cells have similar eNOS and iNOS expression to the parent cell line whereas ZR-PR-LT cells line have much reduced levels of both eNOS and iNOS.
Section snippets
Materials
Anti-eNOS, bovine endothelial cell (599-613) (rabbit) and anti-iNOS mouse macrophage (1131-1144) (rabbit) were obtained from Calbiochem (Nottingham, UK). The StreptABComplex/HRP Duet (mouse/rabbit) was from Dako Ltd (Buckinghamshire, UK). Sulphanilimide and naphthylethylenediamine dihydrochloride were from BDH (Lutterworth, Leicestershire, UK). RU486 was a gift from Excelgen, France. All other chemicals were from Sigma Chemical Company (Poole, UK).
Cell culture
Human mammary epithelial cells (HMEC)
Immunohistochemical staining for eNOS
The presence of cells staining positive for eNOS monoclonal antibody was demonstrated by the appearance of red-stained cells (Fig. 1). One-hundred percent of HMEC (Fig. 1A) and 100% of ZR-75-1 cells (Fig. 1B) stained positive for eNOS. Only 50% of ZR-75-9a1 cells stained positive for eNOS (Fig. 1C) and less than 5% of ZR-PR-LT cells stained positive for eNOS (Fig. 1D).
Immunohistochemical staining for iNOS
The presence of cells staining positive for iNOS monoclonal antibody was demonstrated by the appearance of red-stained cells (
Discussion
The role of nitric oxide in cancer biology is at present poorly understood as it has both pro- and anti- tumor functions. We have previously demonstrated the presence of the l-arginine/nitric oxide pathway in ZR-75-1 human breast cancer cells [17]. The aim of the current study was to investigate expression of both eNOS and iNOS in normal human mammary epithelial cells and in two more malignant variant breast cancer cell lines, namely the estrogen-independent ZR-PR-LT cell line [16] and the
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