Inducible Nitric Oxide Synthase Expression, Apoptosis, and Angiogenesis in in Situ and Invasive Breast Carcinomas¹

Nitric oxide (NO), a diatomic radical, plays a variety of regulatory functions in vivo. It has diverse physiological and pathophysiological roles as a vasodilator, neurotransmitter,antimicrobial effector molecule, and immunomodulator (1). NO is synthesized from the amino acid l-arginine by the NOS³(2). As a free radical, NO is highly reactive and reacts in biological systems with other free radicals, molecular oxygen, and heavy metals (3). The biological effects of NO are mainly mediated by the products of different NO metabolites (3).

There are three isoforms of NOS: iNOS (NOS2), eNOS (NOS3), and nNOS(NOS1; Ref. 1). Each isoform is the product of a distinct gene (4). eNOS and nNOS are constitutive,calmodulin-dependent enzymes (cNOS; Ref. 4). iNOS is expressed in macrophages, neutrophils, endothelial cells,hepatocytes, cardiac myocytes, chondrocytes, and many other cell types (5). It is induced most importantly by cytokines and can generate locally high concentrations of NO for prolonged periods of time (4, 6). Calcium independence of iNOS has been questioned with the report of iNOS enzymatic activity dependent on intracellular fluxes of calcium and binding of calmodulin; but in general, iNOS is calcium-independent (6).

The genotoxicity of NO is attributable to its reaction with either oxygen or superoxide (7). The intracellular NO quickly forms nitrite and nitrate, S-nitroso-thiols, or peroxynitrite (3). NO metabolites can mediate genotoxicity and influence the initiation of cancer by a variety of mechanisms. For instance, NO causes DNA damage by nitrosative deamination, DNA strand breakage, or DNA modification (e.g., nitration) by peroxynitrite (3, 7). These reactions may also be associated with the activation of carcinogenic nitrosamines, initiation of apoptosis and inhibition of DNA repair enzymes, or lipid peroxidation-induced DNA damage (6, 7).

The effects of NO can be tumor promoting or tumor suppressing. High concentrations of NO can be cytotoxic, whereas low concentration may even protect some cell types from damage and apoptosis (3). During the initiation of tumor growth, natural killer cells and macrophages kill tumor cells by a NO-mediated mechanism (5). However, NO may also suppress the antitumor defense,promote tumor angiogenesis and blood flow in the tumor neovasculature,and enhance tumor growth, invasion, and metastasis (5).

NO exhibits contradictory effects on the regulation of apoptosis. It has been demonstrated to be both pro- and antiapoptotic. The proapoptotic effects appear to be linked to the pathophysiological condition where the induction of iNOS is associated with high concentrations of reactive nitrogen metabolites (8). Cell protection is associated with the up-regulation of several protective proteins, such as cyclo-oxygenase-2 or heme-ozygenase-1 (9). Typical findings in NO-mediated apoptosis include accumulation of tumor suppressor protein p53, caspase activation,chromatin condensation, and DNA fragmentation (3, 8, 9).

In human primary breast cancers, relatively high iNOS immunoreactivity has been noted in stromal cells, and the presence of stromal reactivity appears to correlate with tumor grade (10). However, in another study, iNOS positivity was predominantly found in the tumor cells associating positively with the presence of axillary lymph node metastasis (11). In breast cancer, a high extent of apoptosis is usually associated with poor prognosis, and more apoptosis is seen in tumors of high grade (12, 13, 14). The expression of iNOS in relation of apoptosis has not been previously studied in different types of breast cancers. In this study, we evaluated the immunohistochemical distribution of iNOS in in situ and invasive breast cancers and its relation to the apoptotic index, as determined by the terminal deoxynucleotidyl transferase-mediated nick end labeling method. The sections were also studied immunohistochemically for vascular density using FVIII antibody to see whether changes in the expression of iNOS could influence tumor angiogenesis.

A total of 111 breast lesions consisting of 43 in situductal carcinomas, 56 ductal invasive carcinomas, 10 lobular invasive carcinomas, one mucinous, and one medullary carcinoma were collected from the files of the Department of Pathology, University of Oulu. In situ carcinomas consisted of 15 low-grade (5 papillary, 3 solid, and 7 cribriform), 13 intermediate (1 papillary, 8 solid, and 4 cribriform), and 15 high-grade (1 cribriform and 14 comedo-type)lesions. In invasive ductal carcinomas, there were 9 well-differentiated (grade I), 24 moderately differentiated (grade II),and 23 poorly differentiated (grade III) tumors. The material had been fixed in neutral formalin and embedded in paraffin. The diagnosis of all of the cases were based on light microscopic examination by the conventional H&E stain (15, 16). The grading of the ductal invasive carcinomas were made according to Elston and Ellis (16), and the grades of the in situ lesions were made by Holland et al. (17).

The TNM classification was available in 104 cases. There were 43 T_IS, 45 T_1–2, and 16 T_3–4 tumors. Thirty-four cases contained nodal metastases (N_1–3), and distant metastases(M₁) were present in four cases. Mean follow-up time was 5.8 years (range, 0–20 years). In 32 cases, the cancer relapsed. Information about estrogen and progesterone receptors were available for 63 cases. Values >10 fmol were considered positive.

Five-μm paraffin sections were cut from the specimens and placed on SuperFrostPlus glass slides (Menzel-gläser, Germany). Immunostainings with iNOS antibodies were performed as follows. Paraffin sections were soaked in xylene to remove paraffin and rehydrated in graded alcohol series. The sections were heated in a microwave oven in 10 mm citric acid monohydrate (pH 6.0)for 10 min and then cooled properly at room temperature. The endogenous peroxidase was consumed by immersing the sections in 3% hydrogen peroxide in absolute methanol for 15 min. Two different primary antibodies were used: a rabbit polyclonal (dilution, 1:200;Santa Cruz Biotechnology) and a mouse monoclonal (dilution, 1:60;Transduction Laboratories, Lexington) iNOS antibody, which were both incubated for 60 min at room temperature. With these two antibodies, Histostain-PLUS BULK KIT (Zymed Laboratories Inc., South San Francisco, CA) was used. The color was developed by aminoethyl carbazole substrate solution (Zymed Laboratories Inc.). The sections were counterstained in Meyer’s hematoxylin followed by 2% ammonia water handling, after which the slides were mounted with Immu-Mount(Shandon, Pittsburgh, PA).

Negative control slides were prepared from the same tissue blocks. Instead of using the primary antibody, we used PBS. In addition, in seven cases with clear polyclonal iNOS positivity, an absorption test was conducted. Before application to the slides, antibody binding to antigen was neutralized by 2-h preabsorption at room temperature with a 5-fold excess of blocking peptide (Santa Cruz Biotechnology, Inc.) to polyclonal iNOS antibody. Macrophages and neutrophils labeled very strongly (++++) in every slide, and they served as an internal positive control for the immunostaining.

The intensity of iNOS immunostainings was evaluated by dividing the cytoplasmic staining reaction in four groups: 1 = weak; 2 =moderate; 3 = strong; and 4 = very strong cytoplasmic staining intensity. The quantity of immunostainings were evaluated as follows: 0 = no positive immunostaining, 1 = <25%; 2 =25–50%; 3 = 50–75%; and 4 = >75% of tumor cells showing cytoplasmic positivity. A combined score for iNOS immunostainings,based on both qualitative and quantitative immunostaining, was composed by adding the qualitative to the quantitative score. This sum score was then divided in five groups as follows: − = 0; + = 1–2; ++ = 3–4;+++ = 5–6; and ++++ = 7–8.

iNOS staining in stromal cells was evaluated with ×40 objective semiquantitatively and divided in three groups as follows: weak 1 = 0–2 positive stromal cells/HPF; moderate 2 = <10 positive stromal cells/HPF; and strong 3 = >10 positive stromal cells/HPF.

A combined score for iNOS staining in tumor and stromal cells was also calculated. This sum score was divided in four groups as follows:1 = 1; 2 = 2–3; 3 = 4–5; and 4 = 6–7.

For FVIII-related antigen, the immunostaining was performed as follows. The sections were dewaxed in xylene and rehydrated in graded alcohol series. For enzyme predigestion of formalin-fixed tissue, the sections were incubated for 30 min at 37°C in 0.04% pepsin (Sigma Chemical Co., St. Louis, MO) in 0.01 m HCl. The endogenous peroxidase activity was consumed by immersing the sections with 3%hydrogen peroxide in absolute methanol. Nonspecific binding was blocked by incubating the slides in 20% FCS in PBS for 20 min. The primary polyclonal antibody for factor VIII (DAKO A/S, Glostrup, Denmark) was diluted 1:250 in PBS and incubated 30 min at room temperature. Then a biotinylated secondary antirabbit antibody (DAKO A/S) diluted 1:300 in PBS was applied on the sections for 30 min, followed by the avidin-biotin-peroxidase complex (DAKO A/S). The color was developed by diaminobenzidine, after which the sections were lightly counterstained with hematoxylin and mounted with Eukitt (Kindler, Freiburg, Germany).

As a positive control, we used slides from a highly vascularized tumor. Negative controls consisted of PBS instead of the primary antibody.

CMVDs were counted from an average of six HPFs with ×40 objective. Any endothelial-cell cluster consisting of two or more cells was considered a single, countable microvessel. In in situ carcinomas, two distinct vascular patterns could be seen: a diffuse increase of stromal vascularity between ducts and a dense rim of microvessels adjacent to ducts. At first, both of them were counted together. The mean of six counts was calculated and used in statistical analysis. Also, the periductal vessel density (1/mm) was evaluated separately. The periductal vessels from five round neoplastic ducts were calculated. This sum was then divided by the sum of the measures around these ducts evaluated using the radius of the ducts (2πr), which was measured by an ocular micrometer.

To detect apoptotic cells, in situ labeling of the 3′ ends of the DNA fragments generated by apoptosis associated endonucleases was used. The 3′ end labeling of DNA was performed using the ApopTag in situ apoptosis detection kit (Oncor, Gaithersburg, MD)with a few modifications as previously described (18, 19). A positive control consisted of a lymph node with follicular hyperplasia. The sections, after been dewaxed in xylene and rehydrated in ethanol, were incubated in 20 μg/ml Proteinase K (Boehringer Mannheim GmbH, Mannheim, Germany) at room temperature for 15 min. The endogenous peroxidase activity was blocked by incubating the slides in 3% hydrogen peroxide in PBS (pH 7.2). The slides were then treated with terminal transferase enzyme and digoxigenin-labeled nucleotides,after which antidigoxigenin-peroxidase solution was applied on the slides. The color was developed with diaminobenzidine, after which the slides were lightly counterstained with hematoxylin and mounted with Eukitt (Kindler).

Cells were defined as apoptotic if the whole nuclear area of the cell labeled positively. Apoptotic bodies were defined as small positively labeled globular bodies in the cytoplasm of the tumor cells, which could be found either singly or in groups. To estimate the apoptotic index (the percentage of apoptotic events in a given area), apoptotic cells and bodies were counted from 10 HPFs with ×40 objective, and this figure was divided by the number of tumor cells in the same HPFs.

To test the specificity of the two iNOS antibodies, immunoblotting analysis using mouse macrophage lysate (Transduction Laboratories) was performed. According to the manufacturer, the lysate was prepared from the RAW 264.7 (ATCC TIB71) cell line. These cells were established from an ascites tumor derived from a male mouse, which was injected with the Abelson leukemia virus. Mouse macrophage cells were stimulated with IFNγ and lipopolysaccharide for 12 h. The control macrophages were mixed with the electrophoresis sample buffer and boiled for 5 min at 95°C. Seventy-five μg of cell protein were applied to a 12%SDS-polyacrylamide gel (20). The gel was electrophoresed for 2.0 h (80 V) at room temperature, and the protein was transferred onto Hybond enhanced chemiluminescence nitrocellulose membranes (Amersham, Arlington Hights, IL) in a Mini-PROTEAN II Cell(Bio-Rad, Hercules, CA). The blotted membrane was incubated with the poly- and monoclonal antibodies to iNOS (dilutions, 1:2000 for both antibodies) followed by treatment with secondary antimouse and antirabbit antibodies (dilutions, 1:2000 for both secondary antibodies;Jackson Immunoresearch Laboratories) conjugated to horseradish peroxidase. The proteins were detected by enhanced chemiluminescence system (Amersham). Cell protein was measured using the Bio-Rad protein assay (Bio-Rad; Ref. 21).

SPSS for Windows (Chicago, IL) was used for statistical analysis. The significance of associations were determined using Fisher’s exact probability test, correlation analysis, and the two-tailed ttest. Survival was analyzed by applying the Kaplan-Meier method with log-rank analysis. Probability values ≤0.05 were considered significant.

The results of the study are compiled in Table 1. Strong iNOS expression could be seen in stromal macrophages and neutrophils. Also, stromal fibroblasts and endothelial cells often expressed cytoplasmic positivity for iNOS. In benign nonneoplastic breast epithelial cells, inconsistent expression of iNOS could be seen both in the ductal and acinar structures (Fig. 1).

In the whole material, 51 (45.9%) cases showed no staining for iNOS in the tumor cells, 49 (44.1%) stained weakly (+), 10 (9.0%) stained moderately (++), and one (0.9%) stained strongly (+++). None of the breast tumors labeled very strongly (++++) with the antibody. The number of positive stromal cells was small in 41 (36.9%), moderate in 46 (41.4%), and high in 24 (21.6%) cases. There was a clear association between tumor cell positivity and high number of positive stromal cells (P = 0.017). The sum scores for iNOS immunoreactivity were 1 in 24 (21.6%), 2 in 62 (55.9%), and 3 in 25(22.5%) cases. None of the cases reached a sum score 4.

The distribution of iNOS expression in tumor and stromal cells of in situ carcinomas can be seen in Table 1 (Fig. 2 A). The number of iNOS-positive cases increased from grade I to III (20.0%, 46.2%, and 73.3%). There were significantly less iNOS-positive cases in grade I than in grade II-III in situ lesions (P =0.01) and in grade I-II than in grade III in situ lesions(P = 0.01). No significant differences were found when comparing iNOS positivity in stromal cells or the sum scores in different grades of in situ carcinomas (data not shown).

iNOS expression in tumor and stromal cells of invasive carcinomas are compiled in Table 1 (Fig. 3,Aand Fig. 4 A). No significant difference was found in iNOS positivity in tumor and/or stromal cells between different grades in invasive ductal carcinomas (data not shown). Invasive ductal carcinomas contained more cases with iNOS-positive tumor cells than invasive lobular carcinomas (64.3% and 30.0%, respectively; P = 0.04).

iNOS positivity was more often found in tumor cells of invasive ductal carcinomas than in in situ carcinomas (P =0.05; Table 2). Also, the number of positive stromal cells and sum scores were higher in invasive ductal carcinomas (P = 0.03 and P = 0.004,respectively). All different invasive carcinomas had more cases with a very high number of iNOS-positive stromal cells than in situcarcinomas (P = 0.03), and they had also higher sum scores (P = 0.006).

All PBS control slides were negative. iNOS immunoreactivity was completely abolished in all slides where the antibody was pretreated with the blocking peptide in the absorption experiment (Fig. 3,C and Fig. 4 C).

The apoptotic indices are shown in Table 1. The mean apoptotic index was 1.16 ± 1.14%, and the median was 0.63%. Low-grade in situ lesions showed a significantly lower extent of apoptosis(0.29 ± 0.23%) than intermediate and high-grade lesions(0.93 ± 0.88%; P = 0.009). Similarly, low- and intermediate-grade lesions showed a lower extent of apoptosis(0.55 ± 0.67%) than high-grade in situ lesions(1.00 ± 0.92%; P = 0.07). In different grades of ductal invasive carcinomas, the apoptotic indices increased with the tumor grade: grade I (0.71 ± 0.76%) and grade II-III (1.81 ± 1.28%; P = 0.02) or grade I-II (1.28 ±1.30%) and grade III (2.14 ± 1.07%; P = 0.01). There were also significant differences in apoptotic indices between invasive ductal (1.64 ± 1.27%) and in situ carcinomas(0.70 ± 0.79%; P < 0.001) and between in situ lesions and all invasive carcinomas (1.45 ± 1.24%; P = 0.001).

The results of CMVDs detected by FVIII-related antigen are shown in Table 1. The mean CMVD was 14.2 ± 7.1/HPF, and the median was 12.8/HPF. There were no significant differences in vascular density between in situ or invasive carcinomas or between different grades of the tumors (data not shown).

In in situ carcinomas, the average periductal microvessel density was 10.8 ± 5.8/mm (range, 0.8–31.2/ mm; median,10.15/mm). The densities in different grades of in situcarcinomas were: low grade, 9.0 ± 4.9/mm; intermediate, 12.0 ± 7.5/mm; and high grade, 10.9 ± 4.8/mm. There were no significant differences comparing periductal microvessel densities between grade I and grades II-III or between grades I-II and grade III(P = 0.22 and P = 0.79, respectively).

In the whole material, tumors with high sum scores (>1) for iNOS had a higher apoptotic index (≥0.63%; Fisher’s exact test, P = 0.02; Table 3). Moderate or high number of iNOS-positive stromal cells associated also with higher apoptotic index (P = 0.03), but iNOS positivity in tumor cells alone did not (P = 0.12).

There was a significant association between iNOS positivity in tumor cells and a high (≥12.8) CMVD value (P = 0.04). Also,a moderate or high number of positive stromal cells associated with high CMVD index alone (P = 0.05) and as the sum with iNOS positivity in tumor cells (P = 0.02; Table 4). The periductal microvessel density tended to be higher in iNOS-positive (12.3 ± 6.3/mm) than in iNOS-negative (9.1 ± 5.0/mm) cases (P = 0.08).

There was no difference in iNOS positivity in tumor cells between invasive (T_1–4) and in situ(T_IS) carcinomas (P = 0.14), but in invasive carcinomas, there were more cases with a high number of positive stromal cells (P = 0.05). Also, the sum of iNOS positivity in tumor and stromal cells associated clearly with invasive tumors (P = 0.004). A high number of iNOS-positive stromal cells associated with nodal and/or distant metastasis (P = 0.05). The iNOS positivity in tumor cells or the sum of iNOS positivity in tumor and stromal cells did not correlate with nodal and/or distant metastases (data not shown).

iNOS positivity in tumor or stromal cells did not associate with survival, estrogen, or progesterone receptor positivity (data not shown). However, the sum of iNOS positivity in tumor and stromal cells correlated with the progesterone receptor positivity (P = 0.02).

To study the reliability of polyclonal iNOS antibody, we evaluated iNOS activation with a monoclonal iNOS antibody from 53 samples. According to the results, there was a strong positive correlation between iNOS positivities with the polyclonal and monoclonal iNOS antibodies(P = 0.002; Fig. 3,B and Fig. 4 B).

To further test the reliability of the polyclonal and monoclonal iNOS antibodies, an immunoblot analysis with control macrophages was performed with both of them. Positive bands corresponding to the M_r 130,000 of iNOS protein could be detected with both antibodies (Fig. 5).

This study was undertaken to investigate the expression of iNOS in in situ and invasive carcinomas of the breast. Because NO is known to influence apoptosis and angiogenesis, we also studied the extent of apoptosis and vascular density in the breast lesions.

A previous study has demonstrated that in human breast tumors, iNOS is mainly expressed in stromal cells and not in tumor cells and that its stromal presence correlates with tumor grade (10). However, a study with ZR-75–1 human breast cancer cells revealed that these cells contain iNOS and spontaneously produce NO (22). Although we also observed strong expression of iNOS in stromal macrophages and neutrophils in our study, a proportion of tumor cells also displayed clear positivity. In fact, 58.8% of all invasive and 46.5% of in situ lesions displayed some iNOS positivity in a proportion of the tumor cells. The results thus suggest that, in addition to stromal macrophages and neutrophils, breast carcinoma cells also contain detectable levels of iNOS and are thus capable of producing NO. In fact, the results of a recent study by Dueñas-Gonzales et al. (11) are in keeping with our results. To substantiate the findings, we immunostained a part of the lesions with a monoclonal iNOS antibody. Immunostaining with this antibody also showed the presence of iNOS in breast carcinoma tumor cells, and the results were thus consistent with the results obtained by the polyclonal iNOS antibody.

In other types of epithelial tumors, iNOS positivity has been reported in tumor cells of prostate carcinoma (23), gynecological carcinoma (24), colon carcinoma (25, 26), and transitional cell carcinoma of the bladder (27). Recently,strong iNOS synthesis was also discovered in malignant mesothelioma.⁴These results are consistent with our findings and suggest that in addition to other neoplasms, breast carcinoma cells are also able to modulate NO synthesis.

In in situ lesions of the breast, there were significantly more cases with iNOS-positive tumor cells in high-grade than in low-grade tumors. iNOS positivity in tumor cells increased in ductal lesions from in situ carcinomas to invasive. Invasive carcinomas had also more cases with a very high number of iNOS-positive stromal cells than in situ carcinomas. These results suggest that there is an up-regulation of iNOS positivity along with the biological aggressiveness of the breast lesions. Increased iNOS activity has been positively correlated with the degree of malignancy also in gynecological tumors (24). In colon carcinomas,the expression of iNOS remains controversial while both decreased (25, 28) and increased (26) expression of iNOS has been reported with increasing tumor stage.

NO has been reported to be both pro- and antiapoptotic. It has been shown to inhibit apoptosis in several cell types, including endothelial cells (29), hepatocytes (30), lymphocytes (31), leukocytes (32), and eosinophils (33). NO induces apoptosis in various cells,including macrophages (34), pancreatic β-cells (35), and thymocytes (36). There is also a study suggesting that low concentration of NO inhibits apoptosis, but high concentrations of NO induces apoptosis in human venous endothelial cells (37). The role of NO in apoptosis appears to be cell-type-specific and depends on the NO concentration being produced.

In our material, there was a gradual increase in apoptotic index from low-grade in situ carcinomas to high-grade invasive ductal carcinomas with a coexistent up-regulation of total iNOS. A consequent increased production of NO might thus be one reason for the accelerated apoptosis. There were significantly more cases with a high apoptotic index showing iNOS positivity in tumor and stromal cells than in cases with a low apoptotic index. These results suggests that NO, produced by iNOS in breast tumor and in stromal cells, could be an additional factor participating in the enhancement of apoptosis in them.

There are several reports concerning the role of NO in angiogenesis. In vitro studies have demonstrated that NO donors increase and iNOS inhibitors attenuate DNA synthesis, proliferation, and migration of coronary venular endothelial cells (38). On the other hand, both NO donors and iNOS inhibitors have no effect on the fibroblast-growth-factor-induced proliferation and migration of endothelial cells (38). In vivo studies have shown that NO donors potentiate and iNOS inhibitors attenuate angiogenesis in rabbit (38) and rat cornea (39). Also, iNOS-transfected human colon adenocarcinoma DLD-1 cells had higher vessel density and growth rate in vivo than parental cells (40). In the murine mammary tumor model, the data suggest that NO is a key mediator of C3L5 tumor-induced angiogenesis being reduced in NOS inhibitor-treated mice (41). However, controversial results are also obtained (42, 43, 44). These findings suggest that NO partially mediates angiogenesis and that the involvement of NO is both tissue-and/or growth stimuli-dependent.

We studied tumor angiogenesis with an antibody to FVIII-related antigen and compared vascular density with iNOS expression. Tumors with iNOS positivity in tumor and/or stromal cells had increased vascular densities in the whole material. Also, in in situ carcinomas where vascular densities could also be determined in the vicinity of neoplastic ducts, the vascular densities tended to be higher in iNOS-positive cases. These results suggest that local NO production by iNOS in breast carcinoma cells are able to modulate angiogenesis. NO production by stromal cells enhance this effect even more.

iNOS expression may play a role in human cancer progression. Although a few reports indicate that the presence of NO in tumor cells or their microenvironment is detrimental for tumor-cell survival, a large body of evidence suggests that NO promotes tumor progression (6, 45, 46). In murine mammary adenocarcinoma, increased NO production has been shown to promote tumor-cell invasiveness (47). We tested whether the iNOS positivity in breast cancer influences tumor growth measured by TNM status or patient survival. There were no associations between iNOS positivity in breast tumor cells alone and TNM class. However, there were more cases with iNOS positive tumor and stromal cells within invasive tumors (T_1–4)compared to in situ tumors (T_IS). Also, nodal and distant metastases increased in cases with iNOS-positive stromal cells. These results indicate that in breast tumors NO produced by stromal cells enhance tumor growth, invasiveness,and metastatic ability. A part of this effect might be mediated through an increased angiogenesis caused by iNOS, which, on one hand, would enhance the nutrition of the tumor cells, and on the other hand,through increased vessel density, would make more blood vessels available for tumor cells to invade.

There are no previous in vivo or in vitro studies on iNOS expression and hormone receptors in breast cancer. However,eNOS and nNOS have been shown to be expressed only in estrogen-receptor-positive breast cancer cell lines (48). Studies with other tissues or cell lines have revealed that high amounts of estradiol induce iNOS production in rat aortas (49) and in human umbilical vein endothelial cells (50). However, physiological concentrations of 17β-estradiol inhibit iNOS production in the murine macrophage cell line (51). Also, progesterone has been shown to inhibit iNOS production in murine macrophages (52). In our material, there were no associations between iNOS positivity and estrogen receptor positivity. However, the sum of iNOS positivity in tumor and stromal cells correlated with the progesterone receptor positivity. The results indirectly suggest that hormone receptor status and hormonal stimulus may influence iNOS expression in breast carcinoma cells. What effect certain hormone stimulus eventually has seems to be dependent on cell type and hormone concentration.

At the present time, the role of NO in tumor biology is still poorly understood. According to previous reports, NO seems to have a double-edged role in tumor progression, apoptosis, and angiogenesis. iNOS response to hormone stimulus varies also depending on cell type and activity. The effect of iNOS depends on the concentrations of NO being produced and the local environment in different tumor and cell types. Our results show that in addition to stromal cells, iNOS is expressed in neoplastic cells of breast carcinoma. In ductal lesions,the iNOS positivity in tumor cells increased from in situcarcinomas to invasive carcinomas. Invasive carcinomas had also more cases with a very high number of iNOS-positive stromal cells than in situ carcinomas. The results thus show that there is an up-regulation of iNOS positivity along with the biological aggressiveness of the breast lesions. NO produced by iNOS in breast tumor and stromal cells seems to enhance apoptosis. On the other hand,NO production by tumor cells and stromal cells increases tumor vascularization and possibly through this effect also enhance tumor growth and metastatic ability.