Original article | DOI: 10.26402/jpp.2018.6.05

A. KIJ1,2, K. KUS1, M. SMEDA1, A. ZAKRZEWSKA1, B. PRONIEWSKI1, K. MATYJASZCZYK1,2,
A. JASZTAL1, M. STOJAK1, M. WALCZAK1,2, S. CHLOPICKI1,3

DIFFERENTIAL EFFECTS OF NITRIC OXIDE DEFICIENCY ON PRIMARY TUMOUR GROWTH, PULMONARY METASTASIS AND PROSTACYCLIN/THROMBOXANE A2 BALANCE IN ORTHOTOPIC AND INTRAVENOUS MURINE MODELS OF 4T1 BREAST CANCER

1Jagiellonian University, Jagiellonian Centre for Experimental Therapeutics (JCET), Cracow, Poland; 2Jagiellonian University Medical College, Chair and Department of Toxicology, Cracow, Poland; 3Jagiellonian University Medical College, Chair of Pharmacology, Cracow, Poland
The role of nitric oxide (NO) in tumour progression and metastasis is not clear, therefore the present work aimed to better characterise the effects of nitric oxide synthase (NOS) inhibition by L-Nw-nitroarginine methyl ester (L-NAME) on primary tumour growth, pulmonary metastasis, inflammatory state and prostacyclin (PGI2)/thromboxane A2 (TXA2) balance in a 4T1 murine model of breast cancer. To distinguish effects of NO deficiency on disease development, 4T1 cancer cells were administered orthotopically or intravenously to Balb/c mice. The systemic NO bioavailability, pulmonary inflammation and plasma levels of thromboxane B2 (TXB2) and 6-keto-prostaglandin F (6-keto-PGF) were assessed. The study shows that, in the orthotopic model of 4T1 breast cancer, L-NAME hampered primary tumour growth, reduced pulmonary metastases, delayed inflammatory response but did not alter biosynthesis of TXB2 and 6-keto-PGF as well as PGI2/TXA2 ratio in cancer-bearing mice. Interestingly, in the intravenous model of 4T1 breast cancer, NOS inhibition did not influence metastasis nor inflammation, but it increased both TXB2 and 6-keto-PGF biosynthesis without affecting PGI2/TXA2 ratio. In conclusion, in a 4T1 murine model of metastatic breast cancer, NO plays a major role in primary tumour development, while NO is not the key mediator of cancer cell extravasation to the lungs. Furthermore, NO-deficiency activates a PGI2-dependent compensatory mechanism only in the intravenous model of 4T1 breast cancer.
Key words:
breast cancer, nitric oxide, nitric oxide synthase inhibition, systemic inflammation, pulmonary metastasis, prostacyclin, thromboxane A2, cancer cells

INTRODUCTION

Cancer diseases represent the major cause of morbidity and mortality nowadays, with breast cancer the most common cancer type in female patients (1-2), which usually forms metastases in distant organs such as lungs, liver, bones and brain. Metastasising tumour cells interact with the host vascular endothelial cells, leading to alternations in the haemostatic system, disruption of vascular endothelium and finally to their dissemination to distant organs (3-4). Endothelial cell-derived vasoprotective agents, including nitric oxide (NO) and prostacyclin (PGI2), play a pivotal role in maintaining haemostasis. However, many perturbations associated with development of inflammatory state and homeostasis disturbance force endothelial cells to produce a plethora of prothrombotic molecules that promote interaction of platelets and leucocytes with the vessel wall (5). The imbalance between prothrombotic platelet-derived thromboxane A2 (TXA2) and its physiological antagonists such as PGI2 and NO can result in the development of vascular endothelial dysfunction, which facilitates the advancement of not only cardiovascular diseases, but also platelet activation (6-7) and progression of metastasis (8).

It is widely known that platelets actively participate in cancer progression, enhancing tumour angiogenesis (9) and metastasis (10) because they store angiogenic factors that stimulate vessel growth and protect metastasising cancer cells from the host immune system in the circulation (11). Platelet-derived TXA2 and endothelial cell-derived PGI2 are metabolites of arachidonic acid biosynthesised via cyclooxygenase 1 and 2 (COX-1 and COX-2)-dependent pathways (12). Based on many studies, it is known that TXA2 promotes cancer cell proliferation, invasion, migration and participates in cancer angiogenesis (13-14), while exogenous PGI2 and its analogues are considered potent anticancer and antimetastatic agents (15-16). NO and PGI2, as strong antiplatelet molecules, can inhibit the negative effect of platelets and suppress the progression of cancer and metastasis through platelet-dependent mechanisms (13, 17). Therefore, the balance in these two oppositely acting prostanoids (i.e. PGI2 and TXA2) is important for maintaining haemostasis, and alterations in PGI2/TXA2 ratio are observed in some cancer types (13).

NO is a product of endogenous oxygenation of L-arginine via three isoforms of nitric oxide synthases (NOS): neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS). Among them, the neuron-derived nNOS and endothelial-derived eNOS are constitutively expressed mainly in endothelial cells and their activity depends on intracellular Ca2+ concentration, while the expression of iNOS, foremost observed in macrophages and neutrophils, is induced by proinflammatory molecules and is not regulated by Ca2+ level (18). The homeostatic NO levels are cytoprotective and mainly responsible for vascular tone regulation, vascular remodelling, inhibition of platelet aggregation, and prevention of leukocyte adhesion to the vessel wall. Alternatively, too high local NO concentration can be cytotoxic and contributes to nitrosative and oxidative stress, deactivation of proteins and DNA damage (18). The role of NO in cancer diseases is controversial, and contradictory results have been reported (i.e. antitumour properties of NO versus its pro-cancer effects) (19). Considering these data, it has been suggested that tumour treatment by modulation of NO level and its bioavailability may be based on two conflicting strategies: induction or inhibition of NO signalling (17, 19, 20).

In order to better understand the role of NO in breast cancer progression in vivo, in the present study, the effects of NO deficiency on primary tumour growth and pulmonary metastasis were evaluated after orthotopic inoculation of 4T1 cancer cells in comparison with the intravenous injection of 4T1 cancer cells. Such an approach was used to discriminate between an orthotopic model allowing to observe effects resulting from primary tumour growth and spontaneous metastasis, and an intravenous model of experimental metastasis without the presence of primary tumour (21). In our study we used the 4T1 mouse model of metastatic breast cancer, which is a well characterized and widely used animal model, relevant to human breast cancer (22). We found that NOS inhibition hampered the growth of primary tumour in the 4T1 orthotopic model, but did not affect metastatic spread in the 4T1 intravenous model. Therefore, we suggest that NO plays a major role in primary tumour development in a 4T1 murine model of metastatic breast cancer, while cancer cell extravasation to the lungs does not seem to be NO-dependent. Interestingly, NO deficiency clearly affected TXA2 and PGI2 biosynthesis in both models, as judged from the concentrations of their stable metabolites thromboxane B2 (TXB2) and 6-keto-prostaglandin F (6-keto-PGF), respectively, with a distinctly activated PGI2-dependent compensatory mechanism only after intravenous injection of 4T1 breast cancer cells.

MATERIALS AND METHODS

Animals

Balb/c female mice (8 – 10-week-old) were obtained from the Centre for Experimental Medicine of the Medical University of Bialystok (Bialystok, Poland). Mice were kept in groups of five or six per cage and maintained in the housing room with controlled temperature and humidity conditions under a light/dark cycle. Animals were given a standard chow diet and tap water ad libitum throughout the experiment and their body mass was monitored once a week. Animals were sacrificed under anaesthesia obtained with intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). Blood was taken from the right ventricle using a syringe equipped with a plastic tip. Heparinised blood was centrifuged (664 × g at 4°C for 12 min) and collected plasma samples were stored at –80°C for further analysis. After blood drawing, lungs, spleen and primary tumours were carefully isolated and weighed. Additionally, lungs were immediately fixed in 4% buffered formalin solution for the metastases counting and evaluation of inflammatory state.

All procedures carried out on animals were approved by the Local Ethical Committee for Experiments on Animals at the Jagiellonian University (Cracow, Poland, permission no: 138/2016 and 91/2017) and performed complying with EU Directive 2010/63/EU for animal experiments.

Mouse models of metastatic 4T1 breast cancer

Metastatic breast cancer was induced in mice by the administration of the mouse mammary carcinoma 4T1 cells cultured from a cell line obtained from the American Type Culture Collection (ATCC, Rockville, Maryland, USA) according to a previously described protocol (23). After 4T1 cell detachment from the culture plate by accutase, they were counted and suspended in Hank’s Balanced Salt Solution (HBSS). In the orthotopic model of breast cancer, 1 × 104 of 4T1 cells/50 µL were inoculated into the right mammary fat pad of mice, whereas in the intravenous model, 7.5 × 104 of 4T1 cells/100 µL were injected into the mouse tail vein. Tumour cells were administered to healthy mice and mice treated with L-Nω-nitroarginine methyl ester (L-NAME). To induce NO deficiency before cancer cell administration, the designated group of mice was treated with L-NAME at the dose of 100 mg/kg/day delivered in drinking water for one week before cancer cell administration and afterwards until their euthanasia. Mice were divided into the following experimental groups: control group (n = 10, healthy mice) and two 4T1 cancer groups (cancer mice and cancer mice treated with L-NAME) investigated in both orthotopic (n = 12) and intravenous (n = 10) models of metastatic 4T1 breast cancer. The experiment was terminated at the time of pulmonary metastases occurrence: in the orthotopic model, mice were sacrificed three and four weeks after 4T1 cell inoculation, while in the intravenous model, mice were euthanised one week after 4T1 cell injection.

Assessment of 4T1 breast cancer progression

In the orthotopic model, the progression of cancer was assessed based on the primary tumour weight (% of body weight) and volume (V) calculated according to the following equation: V = 0.52*long diameter*short diameter (24). The dimensions of solid tumours were evaluated morphometrically using an electronic calliper. After the dissection of the lungs, whole tissue was fixed in 4% buffered formalin solution and the metastatic nodules on the lungs’ surface were manually counted using a magnifying glass.

Measurements of nitric oxide metabolites in plasma and nitric oxide release in aorta

The concentration of nitric oxide metabolites such as nitrite (NO2) and nitrate (NO3) in plasma samples were determined using an HPLC-based system ENO-20 NOx Analyzer (Eicom, Kyoto, Japan) as previously described (6).

Measurements of stimulated NO release from isolated mouse aorta were performed with an EPR spin-trapping technique according to a previously described protocol (23). Briefly, isolated mouse aortas were incubated with Fe(DETC)2 spin trap and calcium ionophore A23187. After incubation, each aorta was frozen in liquid nitrogen. Samples were stored at –80°C until NO-Fe(DETC)2 measurement using EPR spectrometer (EMX Plus, Bruker, Germany). Results were normalised to the weight of wet aorta.

Histological analysis of pulmonary inflammation

Formalin-fixed lungs were embedded in paraffin blocks and three sections of 5 µm were taken from all five lung lobes and stained using the haematoxylin and eosin method (H&E) according to a standard protocol. Samples were photographed at 200 × magnification with an Olympus BX51 light microscope (Olympus Corporation, Tokyo, Japan) avoiding the metastatic lesions. Because the development of local pulmonary inflammation during breast cancer progression and metastasis results in intensification of macrophage infiltration to the lungs and decreases their alveolar area, the rate of pulmonary inflammation was qualitatively assessed based on changes in lung airness.

UPLC-MS/MS quantification of 6-keto-prostaglandin F1a and thromboxane B2

6-keto-PGF and TXB2 were enriched and extracted from the plasma using a liquid-liquid extraction technique (LLE) according to a previously described procedure with slight modifications (6). Purified plasma samples were injected into the UPLC-MS system comprising a UFLC Nexera (Shimadzu, Kyoto, Japan) coupled to a mass spectrometer QTrap 5500 (Sciex, Framingham, Maryland, USA). Analytes were separated on an Acquity UPLC BEH C18 (3.0 × 100 mm, 1.7 µm, Waters, Milford, Maryland, USA) analytical column in gradient elution mode. Mobile phases consisted of 0.1% FA in ACN and 0.1% FA in water (v/v) were delivered at a flow rate of 350 µL/min. The mass spectrometry detection of analytes and their corresponding internal standards was carried out in the negative ion electrospray ionisation, applying the multiple reaction monitoring mode (MRM).

Data processing

All quantitative results were presented as mean ± SEM and plotted using GraphPad Prism 6.02 software (GraphPad Software Inc., La Jolla, California, USA). Data were statistically analysed applying the adequate parametric tests (T or Tukey tests) or non-parametric calculations (Mann-Whitney and Kruskal-Wallis tests) available in Statistica 13.1 (Statistica, Tulsa, Oklahoma, USA). Results with P-values of 0.05 or less were considered statistically significant.

RESULTS

The effects of nitric oxide synthase inhibition on nitric oxide metabolites in plasma and nitric oxide-dependent function in aorta in orthotopic and intravenous models of 4T1 breast cancer

One week treatment with L-NAME, before 4T1 cancer cell administration, effectively inhibited nitric oxide biosynthesis as evidenced by a fall in NO3 plasma concentration (9.14 ± 1.06 µM versus 12.54 ± 1.56 µM in control mice) (Fig. 1B), but did not significantly change the plasma level of NO2 (0.33 ± 0.02 µM versus 0.30 ± 0.02 µM in control mice) (Fig. 1A).

L-NAME treatment also significantly inhibited systemic vascular eNOS activity in mice resulted in decrease of stimulated NO release from aorta (15181.28 ± 2976.28 arb/mg of aorta versus 24741.52 ± 2323.41 arb/mg of aorta in control mice) (Fig. 1C) without changing the basal rate of NO release from lungs (1035.86 ± 149.01 arb/mg of lungs versus 1026.93 ± 211.38 arb/mg of lungs in control mice) (Fig. 1D).

Figure 1
Fig. 1. The effect of nitric oxide synthase inhibition by L-NAME on plasma concentration of nitric oxide metabolites NO2 (A) and NO3 (B) as well as on eNOS-derived NO released from mouse aorta (C) and lung-derived NO (D) measured by EPR spin-trapping technique before cancer cell administration. Data are presented as mean ± SEM. N = 12 for NO metabolite measurements, n = 5 for NO-Fe(DETC)2; * P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; * indicates differences between control animals and treated with L-NAME.

Orthotopic inoculation of 4T1 cancer cells resulted in significantly decreased plasma concentration of NO2 (0.27 ± 0.01 versus 0.72 ± 0.05 µM in control mice) (Fig. 2A) and NO3 (11.72 ± 0.57 versus 15.00 ± 0.76 µM in control mice) (Fig. 2B) three weeks after cancer development, but not four weeks after cancer cell administration (0.61 ± 0.01 for NO2 and 14.00 ± 1.09 µM for NO3). The production of NO by aorta was not altered three weeks after tumour cell inoculation (20471.13 ± 5555.13 versus 16944.42 ± 970.11 arb/mg of aorta in control mice), however aorta NO release was elevated four weeks after tumour development (26062.05 ± 2695.36 arb/mg of aorta) (Fig. 2C). Treatment with L-NAME did not additionally decrease plasma concentration of NO2 (0.24 ± 0.02 and 0.50 ± 0.05 µM for three and four weeks after cancer cell inoculation, respectively) (Fig. 2A), but markedly reduced plasma NO3 (7.22 ± 0.69 and 7.31 ± 1.06 µM for three and four weeks after cancer cell inoculation, respectively) (Fig. 2B) compared with non-treated cancer-bearing mice. Accordingly, aorta NO production was also decreased by L-NAME in cancer-bearing mice (8660.20 ± 868.31 and 10762.87 ± 1069.92 arb/mg of aorta for three and four weeks after cancer cell inoculation, respectively) (Fig. 2C).

Intravenous injection of 4T1 cancer cells directly into circulation resulted in significantly reduced plasma concentration of NO2 (0.16 ± 0.01 versus 0.57 ± 0.05 µM in control mice) (Fig. 2D), but did not affect plasma concentration of NO3 (14.29 ± 1.64 versus 14.79 ± 0.86 µM in control mice) (Fig. 2E) as well as aorta NO production (24849.74 ± 2975.29 versus 24415.60 ± 2123.10 arb/mg of aorta in control mice) (Fig. 2F) one week after cancer cell injection. In the animals treated with L-NAME, plasma concentration of NO2 was not altered (0.13 ± 0.01 µM) (Fig. 2D), but plasma concentration of NO3 was significantly reduced (8.96 ± 0.87 µM) (Fig. 2E) compared to untreated breast cancer-bearing mice. L-NAME also decreased NO production from aorta (3996.74 ± 538.77 arb/mg of aorta) (Fig. 2F) comparing to 4T1 cancer-bearing mice.

Figure 2
Fig. 2. The effect of L-NAME on NO bioavailability in mouse models of 4T1 breast cancer. Changes in plasma concentration of nitric oxide metabolites NO2 (A, D) and NO3 (B, E) and release of eNOS-derived NO from mouse aorta measured by EPR spin-trapping technique (C, F) in orthotopic and intravenous models of 4T1 breast cancer are presented. In the orthotopic model, measurements for control animals were averaged including the period of three and four weeks. Data are shown as mean ± SEM and were considered statistically significant at *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. N = 12 for the orthotopic model, n = 5 in the orthotopic model for NO-Fe(DETC)2, n = 10 for the intravenous model. * indicates differences between control animals and 4T1 or 4T1 + L-NAME cancer groups, # indicates differences between 4T1 and 4T1 + L-NAME cancer groups.

The effects of nitric oxide synthase inhibition on cancer progression, pulmonary metastasis and inflammatory response in orthotopic and intravenous models of 4T1 breast cancer

In the orthotopic model of 4T1 breast cancer, L-NAME markedly reduced the weight of primary tumour (8.64 ± 1.58 versus 14.00 ± 1.19% b.w. in 4T1 cancer mice) (Fig. 3A) as well as tumour volume (1024.0 ± 88.8 versus 1432.0 ± 183.1 mm3 in 4T1 cancer mice) (Fig. 3B) four weeks after cancer cell inoculation. Accordingly, the number of lung metastases was also significantly lower after NOS inhibition with L-NAME (27 ± 7 versus 50 ± 7 in 4T1 cancer mice) (Fig. 3C) with concomitant lower lung weight (1.21 ± 0.07 versus 1.57 ± 0.13% b.w. in 4T1 cancer mice) (Fig. 3D). It is worth noting that the inhibitory effect of L-NAME on primary tumour development tended to be detectable earlier (i.e. three weeks after 4T1 cancer cell inoculation) as evidenced by a significant decrease in tumour volume (323.4 ± 22.8 versus 478.7 ± 34.0 mm3 in 4T1 cancer mice) (Fig. 3B).

In contrast to the orthotopic model, NOS inhibition by L-NAME did not alter metastatic rates in the intravenous model of 4T1 breast cancer – the number of pulmonary metastases (Fig. 3E) and lung weight (Fig. 3F) were comparable irrespective of L-NAME administration.

Figure 3
Fig. 3. The effect of nitric oxide synthase inhibition by L-NAME on the 4T1 breast cancer progression. The growth of primary tumour (A–B) in the orthotopic model and pulmonary metastasis in orthotopic (C–D) and intravenous (E–F) models of 4T1 breast cancer are presented. In the orthotopic model, measurements for control animals were averaged including the period of three and four weeks. Data are shown as mean ± SEM and were considered statistically significant at *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. N = 12 for the orthotopic model for tumour growth evaluation, n = 6 for the orthotopic model for metastasis assessment, n = 10 for the intravenous model. * indicates differences between control animals and 4T1 or 4T1 + L-NAME cancer groups, # indicates differences between 4T1 and 4T1 + L-NAME cancer groups.

In the orthotopic model, 4T1 breast cancer progression was associated with the development of systemic inflammation indicated by gradually progressing splenomegaly from three weeks (1.65 ± 0.10 versus 0.40 ± 0.01% b.w. in control mice) to four weeks after cancer cell inoculation (2.84 ± 0.13% b.w.) (Fig. 4A). L-NAME treatment compromised increasing spleen weight (1.30 ± 0.07 and 2.23 ± 0.14% b.w. three and four weeks after cancer cell inoculation, respectively) (Fig. 4A) compared to non-treated tumour-bearing mice. However, in the intravenous model of 4T1 breast cancer, no changes in spleen weight were detected (Fig. 4B).

Figure 4 Fig. 4. The influence of L-NAME on the development of systemic inflammation in mouse models of 4T1 breast cancer. The progression of systemic inflammation was assessed based on the spleen weight presented as a percent of mouse body weight (% b.w.) in orthotopic (A) and intravenous (B) models of 4T1 breast cancer. In the orthotopic model, measurements for control animals were averaged including the period of three and four weeks. Data are shown as mean ± SEM and were considered statistically significant at *P 0.05, **P 0.01, ***P 0.001. N = 12 for the orthotopic model, n = 10 for the intravenous model. * indicates differences between control animals and 4T1 or 4T1 + L-NAME cancer groups, # indicates differences between 4T1 and 4T1 + L-NAME cancer groups.

In addition, the suppressive effect of L-NAME on local pulmonary inflammation in the orthotopic 4T1 breast cancer model was visualised by better preserved lung airness when compared to breast cancer-bearing mice not treated with L-NAME (Fig. 5). In contrast, in the intravenous model of 4T1 breast cancer, the lung airness in tumour-bearing mice seemed to be comparable to healthy animals and irrespective of L-NAME treatment (Fig. 5).

Figure 5
Fig. 5. The influence of L-NAME on the development of local inflammation in mouse models of 4T1 breast cancer. The progression of pulmonary inflammation was assessed based on the evaluation of H&E stained sections of representative lung samples from each experimental group in orthotopic and intravenous models of 4T1 breast cancer. The increase in the area of lung tissue in place of alveoli indicates the infiltration of macrophages mainly comprising neutrophils, and reflects the development of an inflammatory state.

The effects of nitric oxide synthase inhibition on prostacyclin/thromboxane A2 balance in orthotopic and intravenous models of 4T1 breast cancer

PGI2/TXA2 balance was assessed based on concentrations of their stable metabolites 6-keto-PGF and TXB2, respectively. Inoculation of 4T1 cancer cells into mammary fat pad resulted in a ca. 2.5-fold increased plasma concentration of TXB2 (Fig. 6A) while no changes were observed in the case of 6-keto-PGF (Fig. 6B). L-NAME did not additionally increase TXB2 biosynthesis and did not promote any changes in 6-keto-PGF plasma concentration compared to non-treated tumour-bearing mice. As a consequence, the balance between 6-keto-PGF and TXB2 in the orthotopic model was shifted toward TXB2, which in turn caused a ca. 2-fold decrease in their ratio compared to control mice (Fig. 6C). The above changes could indicate increased TXA2 production in mice orthotopically injected with 4T1 breast cancer cells.

Figure 6
Fig. 6. Alternations in the PGI2/TXA2 system in mouse models of 4T1 breast cancer. The changes in biosynthesis of TXB2 (A, D), 6-keto-PGF (B, E) and their balance (C, F) in orthotopic and intravenous models of 4T1 breast cancer are presented. The ratio of 6-keto-PGF/TXB2 reflects the balance in the PGI2/TXA2 system. In the orthotopic model, measurements for control animals were averaged including the period of three and four weeks. Data are shown as mean ± SEM and were considered statistically significant at *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. N = 12 for orthotopic model, n = 10 for intravenous model * indicates differences between control animals and 4T1 or 4T1 + L-NAME cancer groups, # indicates differences between 4T1 and 4T1 + L-NAME cancer groups.

After intravenous injection of 4T1 breast cancer cells, TXB2 concentration was only slightly increased, while L-NAME treatment triggered a significant increase in TXB2 biosynthesis (ca. 2.5-fold higher versus control mice) (Fig. 6D). In contrast to the orthotopic model, L-NAME also induced elevation of 6-keto-PGF plasma concentration (Fig. 6E). Therefore, 6-keto-PGF/TXB2 ratio in groups of cancer-bearing mice was comparable to healthy control (Fig. 6F).

Figure 7
Fig. 7. Scheme summarising the major differences in the effect of nitric oxide synthase inhibition by L-NAME in orthotopic and intravenous 4T1 cancer cell administration. In the model of orthotopic inoculation of 4T1 cancer cells into the mouse mammary fat pad, L-NAME slowed the growth of primary tumour, reduced the pulmonary metastasis and delayed the systemic and local inflammatory response without changing the decreased PGI2/TXA2 ratio. In the model of the intravenous injection of 4T1 cancer cells into mouse tail vein, L-NAME did not influence the number of metastases in lungs and inflammatory response, but increased TXA2 biosynthesis, with simultaneous activation of a PGI2-dependent compensatory mechanism keeping the PGI2/TXA2 balance unchanged. (↑) indicates an increase, (↓) indicates a decrease and (NC) indicates no changes in measured parameters.

DISCUSSION

Comparing effects of NOS inhibition on the progression of 4T1 breast cancer and pulmonary metastasis in orthotopic and intravenous mouse models revealed that NO could strongly support the growth of primary tumour and, thus, control the rates of spontaneous metastasis. However, in the absence of primary tumour, the role of NO in the process of extravasation of cancer cells injected intravenously (experimental metastasis) seemed to be not so important (Figs. 3 and 7). Moreover, in mice with orthotopically inoculated 4T1 cancer cells, NOS inhibition also compromised systemic (Fig. 4A) and local (Fig. 5) inflammatory response, but did not evoke any significant changes in already decreased PGI2/TXA2 ratio, as reflected by 6-keto-PGF/TXB2 ratio (Fig. 6C). On the other hand, inflammation rates seemed to be not affected by L-NAME treatment in the intravenous model (Figs. 4B and 5) and neither were the rates of lung metastasis. Interestingly, in this model, L-NAME treatment intensified TXB2 (Fig. 6D) and 6-keto-PGF biosynthesis (Fig. 6E), which resulted in unchanged 6-ketoPGF/TXB2 ratio (Fig. 6F) and, thus, PGI2/TXA2 balance.

As a non-selective NOS inhibitor, L-NAME can inactivate both eNOS and iNOS (25) which, depending on the cancer type, can have discrepant effects on disease progression. The influence of L-NAME on the progression of different types of tumours and metastasis process under in vivo and in vitro conditions has been widely described. Some studies show that NOS inhibitors or NO scavengers decreased tumour growth and angiogenesis in murine melanomas (26) and breast tumours (27) as well as reduced vascular permeability in murine sarcomas (28) or breast cancers (29). Furthermore, L-NAME can be also responsible for a decrease in tumour blood flow (29-32) facilitating leukocyte-endothelial interaction (28). However, contradictory reports revealed that reduced bioavailability of NO promoted progression of pancreatic cancer (33) and lung metastasis in ovarian cancer (34) in iNOS-KO mice. These data suggest that the importance of iNOS (and, possibly, other NOS isoforms) in the progression of malignancy depends on cancer type. In the present work, we clearly demonstrated the involvement of NOS in regulation of primary tumour growth in an orthotopic 4T1 breast cancer model, but not in the metastasis process in an intravenous model.

In the case of 4T1 breast cancer, it was shown that 4T1 cells express eNOS (35). Thus, eNOS silencing by L-NAME could not only supress primary tumour angiogenesis (19) but also decrease 4T1 cell migration ability (35), what in turn, could contribute to reduction of spontaneous metastasis rate (Fig. 3C). Inhibition of disease progression in the orthotopic model of 4T1 breast cancer (i.e. primary tumour growth and spontaneous metastasis) might also result from the prolonged L-NAME treatment before inoculation of 4T1 breast cancer cells into mice. Namely, 1-week L-NAME administration to healthy mice evidently induced systemic NO-dependent endothelial dysfunction (Fig. 1C) though systemic NO bioavailability seemed to be preserved as evidenced by maintained NO2 concentrations in the plasma (Fig. 1A), probably at the expense of NO3 (36) (Fig. 1B) at the time 4T1 cancer cells were inoculated to mice. Although 1-week L-NAME treatment did not affect the basal NO production by pulmonary endothelium (Fig. 1D), L-NAME-induced impairment of NO production by endothelial cells of aorta and, possibly, some other large blood vessels could compromise blood supply to the growing tumour and lower the rates of tumour angiogenesis (19, 27). Interestingly, in the orthotopic model of 4T1 breast cancer L-NAME induced only a transient decrease in plasma concentration of NO2 and NO3 (Fig. 2A-2B) suggesting an increase in activity of iNOS isoform at the late stage of metastasis, in response to development of cancer-induced inflammation. Furthermore, slower progression of 4T1 breast cancer and metastasis rate in the orthotopic model after L-NAME administration might also be associated with delayed development of systemic (Fig. 4A) and local (Fig. 5) inflammation. Such an effect of L-NAME was not observed in the intravenous model and, accordingly, metastasis rates were not affected. The results obtained in the present work point out that NO-deficiency influenced not only the tumour growth related with reduced blood flow and angiogenesis, but also decreased the systemic inflammation.

Apart from NO, yet another vasodilatory molecule with endothelium-protective properties is prostacyclin (PGI2). The rates of PGI2 production are reflected by its stable plasma metabolite (6-keto-PGF) while production of antagonistically acting platelet-derived TXA2 is measured in plasma as TXB2 and their balance is disrupted in some cancer types. In endometrial cancer, higher concentrations of TXB2 were observed in cancer tissue compared to healthy tissue while the concentration of 6-keto-PGF was unchanged (37) irrevocably disturbing PGI2/TXA2 balance in the tumour. Moreover, many other studies also showed that the expression of TXA2 synthase (TXS) or TXB2 level in tumour tissue was higher compared to normal tissue whereas the expression of PGI2 synthase (PGI2S) or concentration of its metabolite 6-keto-PGF was unchanged, consequently leading to the lower PGI2/TXA2 ratio in tumours (37, 39). In the 4T1 orthotopic model, we observed similar trends in TXB2 (Fig. 6A) and 6-keto-PGF (Fig. 6B) plasma concentrations resulting in decreased PGI2/TXA2 ratio (Fig. 6C). In the orthotopic model, elevated TXB2 concentrations could originate from higher expression of thromboxane synthase (TXS) in the subset of cells that spontaneously metastasised to the lungs compared with parental 4T1 cells (39). Accounting for non-platelet source of TXB2 in this case, it is not surprising that the compensatory PGI2-dependent mechanism was not activated. Interestingly, although L-NAME affected primary tumour growth (Fig. 3A-3B) and spontaneous metastasis rates (Fig. 3C), it did not alter plasma concentrations of PGI2 and TXA2 metabolites (Fig. 6A-6B) and, thus, did not alter PGI2/TXA2 balance (Fig. 6C). In the intravenous model, L-NAME increased TXB2 production (Fig. 6D) concomitantly with 6-keto-PGF (Fig. 6E), but did not alter the 6-keto-PGF/TXB2 ratio (Fig. 6F) and, thus, the PGI2/TXA2 balance, suggesting the activation of a PGI2-dependent compensatory mechanism.

In conclusion, in this work, we revealed for the first time the differential effects of NOS inhibition on primary tumour (orthotopic 4T1 model) and pulmonary metastasis (intravenous model of 4T1) with simultaneous evaluation of PGI2/TXA2 balance. L-NAME compromised progression of the disease in the orthotopic model by inhibiting primary tumour growth and spontaneous pulmonary metastasis. These events coincided with lower rates of systemic and local inflammation. In the absence of primary tumour, non-selective NOS inhibition had no effects on disease progression as well as inflammation. Therefore, NO-based pharmacotherapeutic strategies should be effective on primary tumour growth, but not succesive on metastasis.

Abbreviations: 6-keto-PGF, 6-keto-prostaglandin F; ACN, acetonitrile; eNOS, endothelial nitric oxide synthase; EPR, electron paramagnetic resonance; FA, formic acid; Fe(DETC)2, diethyldithiocarbamate-Fe(II) complex; H&E, hematoxylin and eosin; HPLC, high performance liquid chromatography; iNOS, inducible nitric oxide synthase; L-NAME, L-Nω-nitroarginine methyl ester; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; NOS, nitric oxide synthase; PGI2, prostacyclin; TXA2, thromboxane A2; TXB2, thromboxane B2; UPLC-MS, ultraperformance liquid chromatography coupled to mass spectrometry.

Acknowledgements: This work was supported by the project METENDOPHA (a grant coordinated by JCET-UJ, No STRATEGMED1/233226/11/NCBR/2015) and partially by the National Science Centre (Poland) (DEC-2015/17/N/NZ7/01044).

The authors would like to thank Krystyna Wandzel and Anna Calusinska for their help in taking care of animals.

Conflict of interests: None declared.

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R e c e i v e d : November 16, 2018
A c c e p t e d : December 30, 2018
Author’s address: Prof. Stefan Chlopicki, Jagiellonian University, Jagiellonian Centre for Experimental Therapeutics (JCET), 14 Bobrzynskiego Street, 30-348 Cracow, Poland. e-mail: stefan.chlopicki@jcet.eu