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.
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.
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).
Nitric oxide synthases (NOS) are a family of three different isoenzymes that convert L-arginine to L-citrulline and generate the free radical gas nitric oxide (NO).1 NO, as an intracellular second messenger, is involved in several biologic regulatory mechanisms, such as vasodilatation, neuronal transmission, smooth muscle relaxation, and cytotoxicity of macrophages and neutrophils. NOS isoenzymes generally are classified into two groups: 1) the calcium dependent constitutive NOS (c-NOS), which mediates neurotransmission (neuronal NOS [n-NOS]) and vasodilatation (endothelial NOS [e-NOS]); and 2) the calcium independent inducible NOS (i-NOS), which continuously releases 1000 times greater amounts of NO and is involved mainly in tumor-induced immunosuppression and macrophage-mediated cytotoxicity.2
The roles of NO and NOS in tumor biology remain poorly understood. It has been known for several years that NO can cause tumor cytostasis, cytotoxicity, and apoptosis and that it can promote angiogenesis combined with an increase in tumor blood flow.3 These findings suggest that NO may have a protective effect as well as a promoter effect, which seems to be dose dependent and biphasic. Low concentrations of NO are proangiogenic and may promote tumor growth; however, at higher concentrations, NO inhibits tumor growth.3
Specific expression of NOS has been reported in various human malignant tissues, such as colon carcinoma,4 prostate carcinoma,5 lung carcinoma,6 head and neck carcinoma,7 malignant melanoma,8 gynecologic carcinomas,9 and various breast carcinoma cell lines.10 Tumor cells express both e-NOS and i-NOS, although at widely different levels. Moreover, expression also is found in tumor-infiltrating macrophages and in the endothelium of vessels surrounding tumors.
Thomsen et al. detected e-NOS expression in tumor cells from eight gynecologic tumors and found a positive correlation with tumor differentiation.9 The same group reported on increased nitrite/nitrate accumulation in a small series of 15 in situ and invasive breast carcinoma tissues compared with benign breast tissue. The level of NOS activity was far below the enzyme activity associated with cytotoxicity and apoptosis. However, on immunohistochemical examination, only tumor infiltrating macrophages, endothelial cells, and myoepithelial cells, but not breast tumor cells, reacted with a polyclonal antibody against e-NOS and a monoclonal antibody against i-NOS.11
The objective of this study was to evaluate the expression of both e-NOS and i-NOS in a larger series of benign, in situ, and invasive breast lesions to demonstrate the activity of NOS and correlate these findings with established histologic and biologic breast carcinoma markers. An attempt also was made to develop a new, better, and more reliable method of measuring NO in tumor tissue.
MATERIALS AND METHODS
Eighty-four randomly chosen patients underwent surgical treatment for a breast lump at the University Hospital in Heidelberg between 1996 and 1997. Among the 41 women with benign lesions, 15 women had proliferative mastopathy, 15 women had nonproliferative mastopathy, 8 women had fibroadenoma, and 3 women had sclerosing adenosis. Seven women were diagnosed with an intraductal carcinoma of the breast. Among another 54 women, 42 women had invasive ductal carcinomas of the breast, 9 women had invasive lobular lesions, and 3 women had invasive tubular carcinoma, with a representative range of stages and grades. The median age at the time of diagnosis in patients with malignant disease was 44 years. All 54 patients underwent breast-conserving surgery or mastectomy and axillary lymph node excision (median, 18 lymph nodes excised; range, 11–33 lymph nodes excised). Twenty-four patients were diagnosed with a tumor measuring < 2 cm in greatest dimension, and 19 patients had tumors measuring 2–5 cm in greatest dimension. There were 2 highly differentiated tumors (Grade 1), 35 moderately differentiated tumors, and 17 poorly differentiated tumors. Thirty-three patients (61%) showed no axillary lymph node involvement, and, in 21 patients, a mean of 5 metastases to axillary lymph nodes were detected (range, 1–22 metastases to axillary lymph nodes).
The DNA index and S-phase fraction were measured by flow cytometry. Estrogen and progesterone receptor contents were determined using a dextran-coated charcoal assay following the European Organization of Research and Treatment of Cancer criteria described previously.12 A DNA index ≥ 1.1 was considered aneuploid, and a steroid receptor content ≥ 10 fmol/mg protein was considered positive.
Tissue blocks were fixed in 4% buffered formalin and embedded in paraffin. Immunohistochemical reactions were performed using antibodies against e-NOS (anti-ECNOS; Transduction Laboratories, Lexington, KY) and i-NOS (donated by J. Pfeilschifter; Department of Pharmacology, University of Frankfurt, Frankfurt, Germany). The polyclonal i-NOS antibody identified a protein of 130 kDa in lysates of smooth muscle cells that were treated with interleukin-1β (100 IE/mL for 24 hours) to stimulate the i-NOS (data not shown). All other products were from Biogenex (San Ramon, CA), unless otherwise indicated. The antihuman e-NOS monoclonal antibody (diluted 1:500; incubated for 60 minutes at room temperature) and rabbit antihuman i-NOS polyclonal antibody (diluted 1:1000; incubated for 60 minutes at room temperature) were used after the tissue sections had been pretreated in the microwave oven (5 times for 3 minutes each in citrate buffer at 700 Watts). Thereafter, the alkaline phosphatase-antialkaline phosphatase method was used.13 The staining procedures were carried out automatically using an automated cell staining system (Optimax Plus; Biogenex).
The staining was evaluated independently by two observers (H.-P.S. and S.L.) using a ranking scale of 0–4 (0 = negative, 4 = strongly positive). Controls included replacement of the primary antibody in the initial incubation with nonimmune mouse immunoglobulin, with normal rabbit serum, with the secondary antibody only, and (as positive controls) with umbilical cord for e-NOS and kidney with interstitial nephritis for i-NOS.
Diethyldithiocarbamate sodium salt trihydrate (DETC), dithiothreitol (DTT), aprotinin, pepstatin A, phenylmethylsulfonyl fluoride, (6R)-5,6,7,8-tetrahydro-biopterin ([6R]-BH4), flavin adenine dinucleotide (FAD), and β-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), were purchased from Sigma Chemical Company (St. Louis, MO). Ferrous sulfate was purchased from Aldrich (Sigma-Aldrich, Saint Quentin-Fallavier, France), and spermin NONOate was purchased from Biomedical Diagnostics (Marne la Vallée, France). The NOS activity was measured on 10 NOS positive tumors.
The production of NO was assayed after the spin trapping of NO on the diethyldithiocarbamate iron complex [Fe(II)(DETC)2] added to the tumor tissue, leading to the paramagnetic spin adduct ([Fe(II)NO(DETC)2]) detectable by electron paramagnetic resonance (EPR). The EPR method reported previously was used with slight modifications.14, 15
The frozen tumor tissue fragments stored at −196 °C were crushed with a mortar and pestle under liquid nitrogen and were incubated in a buffered solution (50 mM N-2-hydroxyethyl-piper-az-ine-N′-2-ethane-sulphonate [HEPES]; 1 mM DTT; 1 mM MgCl2; 5 mg/L pepstatin A; 0.1 mM phenylmethylsulfonyl fluoride; and 3 mg/L aprotinin, pH 7.6) at 37 °C for 30 minutes. Then, the samples (≈ 100 mg of tissue per 0.2 mL of buffered solution) were treated with 0.1 mL of a 1-mM [Fe(II)(DETC)2] solution after the addition of 5 mM NADPH, 5 μM ([6R]-BH4), and 5 μM FAD at 37 °C for 30 minutes. Samples were frozen again in liquid nitrogen for the EPR measurements in calibrated tubes. EPR studies were performed on an MS100 spectrometer (Magnettech Ltd., Berlin, Germany) under the following conditions: temperature, −196 °C; microwave frequency, 9.34 GHz; microwave power, 20 mW; modulation frequency, 100 kHz; modulation amplitude, 0.5 mT; time constant, 100 msec.
Relative [Fe(II)NO(DETC)2] concentrations in the different tissue samples were determined by the comparison of the third component amplitude of the three-line EPR signal.14 After EPR measurements, the tissue samples were dried and weighted (Wds). NO activities were counted as the relative [Fe(II)NO(DETC)2] amount formed during the 30-minute incubation time divided by the weight of the dried sample (Wds). For quantifying the [Fe(II)NO(DETC)2] complex concentration in the tissues, a 15-μM [Fe(II)NO(DETC)2] complex was prepared as a standard according to the following procedure: a solution of 0.5 mM DETC, 0.5 mM NO donor, spermin NONOate, and 15 μM Fe(SO4) were incubated during 2 hours in 15 mM HEPES buffer containing heat-killed yeast for solubilization of the lipofilic complex (100 mg dry yeast per 2 mL were heated at 90 °C during 5 minutes for inhibition of the enzymatic activity). The concentration of the [Fe(II)NO(DETC)2] complex was carried out by comparing the EPR signal intensity in the tumor tissues with the EPR signal intensity of this standard. The NO activity in the tumor tissues was expressed as the concentration of NO produced by 100 mg of dried tissue in 1 minute.
For statistical calculations, semiquantitative estimates of the immunologic staining results were used. NOS expression in malignant tumors was correlated with tumor type, disease stage, and nuclear grade using a Fisher exact test. The Wilcoxon–Mann–Whitney U test was performed for comparisons of nonparametric independent variables. The level of significance for all tests was 5%.
Immunohistochemistry of Breast Tissue Sections
The specificity of the e-NOS antibody has been tested in Western blot analyses with human endothelial cells and showed only one band at 135 kDa. The polyclonal i-NOS antibody identified a protein of 130 kDa in lysates of smooth muscle cells that were treated with interleukin-1β (100 IE/mL for 24 hours) to stimulate i-NOS (data not shown). To test for the specificity of the antibodies, different control experiments were performed: 1) control with normal rabbit serum, 2) initial incubation with the primary antibody was replaced by nonimmune mouse immunoglobulin, and 3) without the primary antibody and using only the secondary antibody. Unspecific staining was not detectable in the controls. In immunohistochemical investigations, e-NOS antiserum stained endothelial cells of small capillaries and epithelial cells of the breast glands (Fig. 1). The incidence of e-NOS expression in epithelial cells differed significantly between benign tissues and malignant tissues (P < 0.00001). Although e-NOS expression was found only in 2 benign tumors (5%; 1 fibroadenoma and 1 proliferative mastopathy), in situ and invasive lesions were positive for e-NOS expression in 56% and 61% of tumors, respectively (Table 1). A high level of coincidence between positive staining for e-NOS in epithelial cells (tumor cells) and endothelial cells (intratumoral capillaries) was found (P = 0.0008). Small capillaries with a positive immunoreaction for e-NOS were located at the invasion front of the tumor and usually were accompanied by lymphocytic reaction, whereas capillaries with no immunoreaction for e-NOS were found more often in the center of tumors.
Table 1. Incidence of Endothelial and Inducible Nitric Oxide Synthase Expression in Benign, In Situ, and Invasive Breast Lesions
None of the benign lesions was positive for i-NOS, whereas i-NOS expression in epithelial cells was found in 67% of in situ carcinomas and in 61% of invasive carcinomas (Table 1). In particular, within inflammatory sites in the close vicinity of infiltrating carcinoma, cells showed strong anti-i-NOS immunoreaction (Fig. 1).
There was a high correlation between i-NOS staining and e-NOS staining (P = 0.00001). Among 54 invasive breast carcinoma lesions, 28 lesions showed positive staining for both i-NOS and e-NOS, whereas 16 invasive breast carcinoma lesions showed no immunoreactivity for either i-NOS or e-NOS. Five tumors exhibited positive staining for either e-NOS or i-NOS. The staining for e-NOS generally was stronger than for i-NOS, and there was less positive Grade 2 or 3 staining for i-NOS than for e-NOS. The e-NOS ranking was as follows: 20 tumors were negative for staining, 12 tumors had Grade 1 staining, 11 tumors had Grade 2 staining, 10 tumors had Grade 3 staining, and only 1 tumor had Grade 4 staining. The i-NOS ranking was as follows: 22 tumors were negative for staining, 18 tumors had Grade 1 staining, 9 tumors had Grade 2 staining, 6 tumors had Grade 3 staining, and 0 tumors had Grade 4 staining.
To examine the activity of e-NOS and i-NOS in breast tumors, we conducted different assays: The classic test for the analysis of NOS activity by converting L-arginin to L-citrullin was performed but did not provide evidence of NO production. A test was needed that could be performed on frozen tumor tissue and that was capable of tracing even small amounts of NO. A sensitive method for detecting NO that had not been used before for measuring NO in tumor tissues was EPR with spin trapping. NO in tumor tissue was detected by EPR spectroscopy at −196 °C after formation of the paramagnetic [Fe(II)NO(DETC)2] complex, as described above. The EPR spectrum of the tumor tissue sample shown in Figure 2 is a superimposition of several paramagnetic species spectra: i.e., iron(II) in HbNO and copper(II) in [Cu(II)(DETC)2] formed during the treatment of the sample. The spectrum of the [Fe(II)NO(DETC)2] complex is characterized by a gm = 2.035 value in frozen solution and a triplet hyperfine structure with a hyperfine splitting constant of about 1.3 mT due to interaction between the unpaired electron and nitrogen in the NO group (in Fig. 2). Quantification of the trapped NO was performed by a double integration of the [Fe(II)NO(DETC)2] spectrum and by comparison of the results with the integral intensity of the EPR signal of a 15-μM [Fe(II)NO(DETC)2] complex solution prepared as described above (see Materials and Methods) and was recorded under the same conditions. Because the second component of the [Cu(II)(DETC)2] complex is superimposed in part with the EPR signal of [Fe(II)NO(DETC)2], the intensity of this copper component (as determined from the integration of the equivalent fourth component) was subtracted. The results of the NO quantification in tumor tissues are shown in Figure 3. The NOS activity in tumor tissues is expressed by the amount of NO produced by 100 mg of dried tissue in 1 minute. The NOS activity of the different tumors ranged from 5.8 ± 1.2 to 28.1 ± 4.5 pmol per minute per 100 mg dried tissue. The e-NOS activity and i-NOS activity were not separated due to the small size of the samples. This method demonstrated that the NOS present in tumors had NO activity and allowed a comparison with NOS expression (Table 2).
Table 2. Nitric Oxide Synthase Activity in Tumor Tissues Measured by Electron Paramagnetic Resonance Spin Trapping and Comparison with Immunohistochemistry Results
Values are shown as the mean ± standard deviation.
11.8 ± 1.2
13.6 ± 0.9
5.8 ± 1.2
11.3 ± 0.5
9.4 ± 2.2
17.2 ± 1.1
28.6 ± 1.3
15.7 ± 3.2
28.0 ± 4.6
10.3 ± 0.5
Correlation of NOS Expression with Clinical and Pathologic Features
After demonstrating the expression and activity of NOS in different breast samples, we wondered whether the expression in breast carcinoma could be correlated with clinical and pathologic parameters. Younger patients (age ≤ 50 years) significantly more often had e-NOS positive tumor cells (P = 0.051), whereas older patients (age > 50 years) tended to have e-NOS negative tumors. Ductal carcinomas significantly more often were e-NOS positive (66.7%) compared with the lobular or tubular carcinoma subtypes (22.2%; P = 0.01). No correlation was found for i-NOS expression. There also was a trend toward a lower median number of involved axillary lymph nodes in e-NOS positive tumors (P = 0.09) and i-NOS positive tumors (P = 0.06). NOS negative tumors showed no spread to lymph nodes in 47.6% of lesions and no spread to lymph nodes in 71.9% of NOS positive tumors (P = 0.09). The e-NOS positive tumors were more frequently highly differentiated or moderately differentiated compared with the e-NOS negative tumors. Cell proliferation was higher in e-NOS negative tumors. The expression of i-NOS was correlated strongly with the expression of e-NOS (P = 0.0008). No correlation was found between e-NOS expression and tumor size, steroid hormonal receptor (estrogen and progesterone) content, or DNA index (Table 3).
Table 3. Correlation of Endothelial and Inducible Nitric Oxide Synthase with Histologic and Biologic Features of 54 Invasive Breast Lesions
This study revealed that c-NOS and e-NOS can be detected by immunohistochemistry predominantly in tumor tissue from malignant breast carcinomas. In contrast, the detection of e-NOS in benign breast lesions is very rare. Using immunohistochemistry, Thomsen et al. detected i-NOS only in peritumoral and intratumoral spindle cells, identified as macrophages, and detected e-NOS in macrophages, myoepithelial cells, and vascular endothelium;11 whereas, in the current study, i-NOS and e-NOS were detected in the cytoplasm of tumor cells. Those authors described significantly higher NOS activity in malignant breast tissue compared with benign breast tissue using the L-arginine/L-citrulline assay.11 Beyond this conventional technique, we determined NOS activity directly with a sensitive method by trapping NO on the bis(diethyldithiocarbamate) iron(II) complex—a more direct procedure. In the small series of 15 invasive breast carcinomas by Thomsen et al., it was suggested that NOS activity was higher in less differentiated tumors. Those first investigations in breast carcinoma were the basis for our investigations. We intended to investigate whether the tumor cell itself produced i-NOS and e-NOS. Furthermore, for the first time, we compared benign lesions, in situ lesions, and invasive lesions of the breast for their expression of e-NOS and c-NOS.
Tschugguel et al. reported on three poorly differentiated breast carcinomas that showed no staining for e-NOS.16 However, those authors found expression of e-NOS in apocrine metaplasia in 21 of 25 benign lesions. Apocrine metaplasia is a benign lesion of human mammary epithelium. The mechanism of development of apocrine differentiation has not been identified. Furthermore, its relation with particular subtypes of mammary carcinoma has not been elucidated to date.17 and the relation between apocrine metaplasia and invasive breast carcinoma remains a matter of controversy.18 However, the results of Tschugguel et al. add an interesting aspect to our experimental results.16 The same group previously demonstrated that 15 human breast carcinoma cell lines expressed c-NOS mRNA, although at a much lower level compared with cultured human umbilical vein endothelial cells.10 A strong correlation was found with the estrogen receptor status of these cells. NOS mRNA was detected only using polymerase chain reaction analysis in estrogen receptor positive cell lines. This coexpression gives rise to the hypothesis that estrogen is a strong enhancer of NO release in (breast) tissue. This view is supported by the findings of several groups that have detected a positive correlation in breast carcinoma tissue between NOS and estrogen receptor status.19, 20 Reveneau et al. investigated 40 malignant lesions and 38 benign lesions of the breast and found a correlation between NOS expression and the progesterone receptor,21 although no correlation with either estrogen receptor status or progesterone receptor status was confirmed. However, premenopausal patients significantly more often had e-NOS positive tumors, which may have been due to the higher estrogen levels in premenopausal women. Earlier experiments on fetal pulmonary artery endothelium have shown that e-NOS gene expression can be stimulated and up-regulated by estrogen and inhibited by simultaneous application of ICI 182.780, a selective estrogen receptor inhibitor that has been used in clinical trials for treating patients with advanced breast carcinoma.22–24
It is a well-known fact that there is a high negative correlation between estrogen receptor status and tumor differentiation in human breast carcinoma. Grade 1 and 2 breast carcinomas more often express estrogen receptors than Grade 3 breast carcinomas.25 This supports the finding in the current study that the expression of NOS is observed more frequently in tumors with a low malignant potential (lymph node negative disease, high differentiation, and low S-phase fraction). Therefore, it seems less likely that NOS activity should be higher in poorly differentiated tumors; this type of correlation was reported by Thomsen et al. and may be explained by the small number of specimens studied by those authors: Like the current study, 50% of all poorly differentiated tumors in their study showed no immunostaining.16
Thomsen et al. also postulated a positive correlation between calcium dependent NOS activity and tumor differentiation in eight gynecologic tumor tissues, although they were unable to detect immunolabeled tumor cells in all but one malignant tissue specimen.9 However, the expression varied widely between the tissues, and there was heterogeneity in the tumor tissue sites (six different ovarian tumor sites, one endometrium specimen, and one mixed mesodermal tumor). In a subset of 16 prostate adenocarcinomas, positive i-NOS immunostaining was detected in all specimens. No differences in the density of immunostaining for i-NOS or in the tumor grade were reported in epithelium, vessels, or macrophages.5
The results reported here show that expressed e-NOS and i-NOS contribute to NO production, which was measured using EPR with spin trapping—a complementary method for investigating free radicals in tumor tissues and detecting small amounts of NO. In this report, we demonstrated for the first time a correlation of immunohistochemical staining for NOS and NO production measured using EPR spectroscopy, which represents a highly sensitive technique. Additional efforts will be required to establish EPR measurements as criteria for determining the malignant potential of breast carcinoma.
We conclude that NOS expression in breast carcinoma may be an early event in carcinogenesis. This view is supported by the high rate of NOS detection in in situ carcinomas. Like the earlier report by Tschugguel et al., a high level of expression of e-NOS was found in apocrine metaplasia in breast tissue.16 It has also been reported that NOS expression in colon carcinoma is lower than that in colorectal polyps.26 Further investigations in larger patient groups and with data on clinical follow-up and outcome are needed to elucidate the role of e-NOS and i-NOS as a prognostic factor in patients with breast carcinoma.