Expression of endothelial and inducible nitric oxide synthase in benign and malignant lesions of the breast and measurement of nitric oxide using electron paramagnetic resonance spectroscopy

Abstract

BACKGROUND Nitric oxide (NO) is present in various human solid tumors and tumor cell lines, and it is believed that NO plays an important role in tumor growth. An increased NO concentration catalyzed by NO synthase (NOS) is cytotoxic and can promote apoptosis. The expression of endothelial NOS (e‐NOS) and induced NOS (i‐NOS) was examined in various breast tumors. METHODS Immunohistochemical staining with a monoclonal antibody (Ab) against e‐NOS and a polyclonal Ab against i‐NOS was performed on paraffin embedded tissues from 41 benign breast lesions, 9 in situ breast lesions, and 54 invasive breast lesions. Functionality was confirmed by detection of NO using spin‐trapping electron paramagnetic resonance (EPR) spectroscopy. RESULTS e‐NOS expression was found in 2 benign lesions (5%; 1 fibroadenoma and 1 proliferative mastopathy), in 5 in situ lesions (56%), and in 33 invasive lesions (61%). None of the benign lesions was positive for i‐NOS, but 6 in situ lesions (67%) and 33 invasive lesions (61%) showed tumor cell staining. In particular, capillaries that were embedded in lymphocytic stroma showed a positive reaction for e‐NOS. The functionality of NOS was demonstrated by direct NO formation using the EPR spin‐trapping method. Tumors that were positive for e‐NOS were observed more often in younger patients (P = 0.05). These tumors more frequently were highly differentiated or moderately differentiated and more often showed invasive ductal subtypes and a lower proliferation rate. Tumors that were positive for both e‐NOS and i‐NOS were more likely to be lymph node negative tumors. Both i‐NOS‐expressing lesions and e‐NOS‐expressing lesions showed strong coexpression (P = 0.00001). CONCLUSIONS NOS is detected predominantly in in situ lesions and invasive breast lesions but rarely in benign lesions. NOS is found more frequently in invasive carcinomas with low malignancy. Using the spin‐trapping EPR method, this study demonstrates direct NO formation in human breast tumors for the first time. Cancer 2002;95:1191–8. © 2002 American Cancer Society. DOI 10.1002/cncr.10817

Part I. Molecular and cellular characterization of high nitric oxide-adapted human breast adenocarcinoma cell lines

“…However, a dichotomy exists between enhanced radiosensitization and conditions where elevated levels of NO and up-regulated NOS are contributors to tumor progression. The relationship between NOS expression and tumor growth is evident in the literature from studies with a variety of cancer types including: (1) human oral squamous cell carcinoma [10,40], (2) gastric carcinoma [41], (3) lymph node metastases, and most significantly for this current work, (4) breast cancer, to a point that it is a universal property for human solid tumors [7,[15][16][17][42][43][44][45].…”

“…In prior work, our laboratory explored expression levels of NOS and nitrotyrosine in various carcinomas [9][10][11][12]. Work by other reserachers has demonstrated that NO is overexpressed in multiple cancer types [13][14][15][16][17][18][19]. In vitro studies have alluded to there being NO concentration levels, which when reached, result in phenotypic changes in tumors [20].…”

The role of nitric oxide in cancer

“…Loibl et al further demonstrated that while none of the benign lesions were positive for iNOS, 67% in situ carcinomas and 61% invasive lesions showed iNOS tumour cell staining. eNOS expression was found in 33 invasive lesions (61%) [22]. Both iNOS expressing lesions and eNOS expressing lesions showed strong co-expression (p=0.0008).…”

“…NO biosynthesis was significantly greater for grade III tumours as compared with grade II in specimens from 10 breast cancer tissues. Recently, three relatively large scale studies [20][21][22], suggested that iNOS is not only expressed in stromal cells and macrophages in the tumour, but also in tumour cells themselves (Tab 1). Reveneau et al reported NOS activity in 27 of 40 tumours studied [20].…”

Promoter polymorphism (−786t>C) in the endothelial nitric oxide synthase gene is associated with risk of sporadic breast cancer in non‐Hispanic white women age younger than 55 years

“…[15][16][17] Expression of the eNOS gene appears to be high in a normal mammary epithelium cell line (i.e., HMEC) but downregulated in breast carcinoma cell lines (i.e., ZR-75-9a1, ZR-PR-LT, and MCF-7 ADR). 18,19 However, there are contrasting results from studies of eNOS expression in breast cancer tissues, [20][21][22][23] most likely because many endogenous and exogenous factors, including genetic variation in the eNOS gene, influence eNOS protein expression levels.…”