Inducible NO synthase inhibits the growth of free tumor cells, but enhances the growth of solid tumors

Manabu Nishikawa1, BaoJun Chang and Masayasu Inoue

Departments of Biochemistry and Molecular Pathology, Osaka City University Medical School, 1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japan

1 To whom correspondence should be addressed Email: nishikawa{at}med.osaka-cu.ac.jp


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
To elucidate the role of nitric oxide (NO) in tumor cell growth in vivo, dynamic aspects of the growth of Ehrlich ascites tumor cells (EATCs) were studied in wild-type (WT) mice and in an inducible strain of NO synthase (iNOS)-deficient (iNOS–/–) mice. Kinetic analysis showed that the rate of free tumor cell growth in the peritoneal cavity was significantly higher in the iNOS–/– mice than in the WT mice. In contrast, EATCs inoculated subcutaneously rapidly grew and formed a solid tumor in WT mice, but failed to grow in iNOS–/– mice. These results clearly indicate that NO generated by iNOS predominantly inhibits the growth of tumor cells in their free form, but enhances the growth of solid tumors.

Abbreviations: EATC, Ehrlich ascites tumor cell; iNOS, inducible type of NO synthase; iNOS–/–, iNOS deficiency; NO, nitric oxide; VEGF, vascular endothelial growth factor; WT, wild-type


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Nitric oxide (NO) plays important roles in the regulation of the circulatory status, in neuronal transmission and in the defense mechanism against pathogens (13). NO generated by activated macrophages has also been postulated to play a key role in host defense against tumor cells (1). We showed previously that peritoneal macrophages in ascites tumor-bearing animals express high levels of inducible NO synthase (iNOS), and release substantial amounts of NO (4), and that NO reversibly inhibits both mitochondrial respiration and ATP synthesis in tumor cells, particularly under physiologically low oxygen tensions (5). Because the oxygen tension in the peritoneal cavity is low, NO released from peritoneal macrophages inhibits mitochondrial respiration in free ascites tumor cells, thereby suppressing ATP-dependent processes such as DNA synthesis, and reducing proliferation of ascites tumor cells. On the other hand, NO up-regulates the expression of vascular endothelial growth factor (VEGF), which leads to increased neovascularization in and around solid tumors (6). The evidence for the roles of NO in tumors has been reported (7). However, the critical difference of NO action in various types of tumor growth in vivo remains to be elucidated. The present work describes the effect of endogenously generated NO on the growth of free ascites tumor cells and solid tumors in vivo.


    Materials and methods
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 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
S-Nitroso-N-acetyl-DL-penicillamine (SNAP) and 2,2'-(hydroxynitrosohydrazono)bis-ethanamine (NOC18), which are both NO donors, were obtained from Dojindo Chemical (Kumamoto, Japan). Rotenone, antimycin A and oligomycin were obtained from Sigma Chemical (St Louis, MO). All other reagents used were of the highest grade commercially available.

Animal experiments
Wild-type (WT) and iNOS-deficient (iNOS–/–) C57BL/6 mice (8) were obtained from Jackson Laboratories (Bar Harbor, ME). All experiments were approved by the Animal Care and Use Committee of Osaka City University Medical School.

Ehrlich ascites tumor cells (EATCs) were inoculated intraperitoneally (106 cells/mouse) and allowed to grow in their free form. On days 4 (0.5 weeks), 7 (1 week), 11 (1.5 weeks) and 14 (2 weeks) after EATC inoculation ascites were obtained from the peritoneal cavity. After centrifugation at 400 g for 5 min, the supernatant fraction (ascites fluid) was removed. The precipitated cells were re-suspended in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and incubated in culture flasks under 5% CO2 and 20% O2. After incubation for 1 h to remove inflammatory cells and fibroblasts, the cell suspension was centrifuged at 400 g for 5 min to collect and count the number of free EATCs.

EATCs were also inoculated subcutaneously (106 cells/mouse) to generate solid tumors. The long diameter of each tumor was measured every 5 days, using calipers.

Analysis of NO metabolites
Concentrations of NO metabolites in plasma and ascites were determined by the method of Green, using high-performance liquid chromatography (9).

Analysis of DNA fragmentation
DNA fragmentation in the EATCs was analyzed by agarose gel electrophoresis. Briefly, EATCs (2 x 106) were incubated at 4°C for 10 min in 100 µl ice-cold lysis buffer (10 mM Tris–HCl pH 7.4, 10 mM EDTA, 0.5% Triton X-100) and centrifuged at 10 000 g for 20 min. The supernatant was incubated with 40 µg RNase A at 37°C for 1 h and subsequently with 40 µg proteinase K for 1 h. DNA was then precipitated with 1 vol of isopropanol and 0.2 vol of 5 M NaCl at –20°C for 12 h, and collected by centrifugation at 10 000 g for 20 min. Pellets were air-dried and dissolved in 10 µl of 10 mM Tris–HCl (pH 7.4) containing 1 mM EDTA. DNA samples thus obtained were subjected to agarose gel (1.6%) electrophoresis at 100 V using 40 mM Tris–acetate buffer containing 1 mM EDTA (pH 8.0) as the running buffer. The gel was stained with 0.1 µg/ml ethidium bromide and visualized under ultraviolet light.

Analysis of cell viability
EATCs were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum in culture flasks, under 5% CO2 and 20% O2 at 37°C in the presence of various regents for 24 h. Cell viability was estimated by microscopic counting of the EATCs that excluded trypan blue.

Western blot analysis
Solid tumors were lysed in a lysis buffer. Lysates (10 µg protein) were subjected to 7.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The electrophoresed proteins were transferred to a nitrocellulose sheet using a Pharmacia semi-dry blot system (2 mA/cm2 for 1 h in 192 mM Tris–glycine buffer). The sheets were incubated in TBS buffer (140 mM NaCl, 50 mM Tris–HCl, pH 7.2) containing 0.1% Tween 20 and 5% low-fat milk powder at 4°C for 12 h. Then, the sheets were incubated with rabbit anti-mouse iNOS antibody (1:1000 in TBS buffer with 0.5% low-fat milk powder) at room temperature for 1 h. The incubated sheets were washed with TBS buffer to minimize non-specific binding of the antibody. After incubation with horseradish peroxidase-conjugated goat anti-rabbit antibody (1:1000 in TBS buffer with 0.5% low-fat milk powder) at room temperature for 1 h and immunoreactive spots were analyzed by ECL (Amersham, Buckinghamshire, UK).

Analysis of gene expression
Total RNA (2 µg) was extracted from EATCs in the solid form by the acid guanidinium–phenol–chloroform method, and was then reverse-transcribed using a reverse transcriptase system. The reaction mixture was used as a template for PCR amplification with the primers 5'-GCGGGCTGCCTCGCAGTC-3' (sense) and 5'-TCACCGCCTTGGCTTGTCAC-3' (antisense), which correspond to base pairs 16–33 and 659–640, respectively, of the mouse VEGF cDNA sequence. RT–PCR products of 644 (VEGF165) and 512 bp (VEGF121), respectively, were obtained. For ß-actin, the primers 5'-TGTGATGGTGGGAATGGGTCAG-3' (sense) and 5'-TTTGATGTCACGCACGATTTCC-3' (antisense) gave rise to a 514-bp RT–PCR product. Amplification was performed over 30 cycles with the following profile for each cycle: 94°C for 1 min, 65°C for 1 min and 72°C for 1.5 min.

Statistics
Statistical analyses of the results were made with Student's t-test.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Growth of free EATCs and levels of NO metabolites
The number of EATCs in the ascites of WT mice increased for 11 days after inoculation, and decreased thereafter (Figure 1A). Interestingly, the rate of EATC growth was much higher in iNOS–/– mice than in WT mice. Figure 1B shows the changes in the volume of ascites fluid after intraperitoneal inoculation of EATCs. The amount of ascites increased with time in both WT and iNOS–/–mice, with the rate of increase being slightly higher in iNOS–/– mice than in WT mice.



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Fig. 1. Free tumor cell growth. EATCs were inoculated intraperitoneally into WT and iNOS-deficient mice (106 cells/mouse) and allowed to grow in their free form. At the indicated times after inoculation, ascites that contained EATCs were obtained from the peritoneal cavity. The number of ascites tumor cells was counted (A) and the ascites volume measured (B). WT, open circles; iNOS-deficient, closed circles. Data are expressed as means ± SD (n = 10). *P < 0.05 versus WT at 1 W, **P < 0.05 versus WT at 1.5 W.

 
To study the metabolism of NO in EATC-bearing animals, changes in the levels of nitrite plus nitrate in ascites and plasma were determined (Figure 2A and B). Plasma levels of the NO metabolites increased markedly in WT mice and only slightly in iNOS–/– mice, and NO levels in ascites increased only in WT animals.



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Fig. 2. NO metabolites in ascites and plasma from mice intraperitoneally inoculated with tumor cells. Mice were inoculated with EATCs, as described in Figure 1. Ascites (A) and plasma (B) were collected and the concentrations of NO metabolites were determined, as described in the text. WT, open bars; iNOS-deficient, closed bars. Data are expressed as means ± SD (n = 10). *P < 0.05 versus iNOS-deficient at 1 W; **P < 0.05 versus WT at 2 W; {dagger}P < 0.05 versus iNOS-deficient at 2 W.

 
DNA fragmentation in free EATCs
To test for apoptosis of EATCs, DNA samples were isolated and analyzed by agarose gel electrophoresis. EATCs obtained from WT mice 2 weeks after inoculation showed marked fragmentation of nuclear DNA. However, no such fragmentation was found in DNA samples in EATCs obtained from iNOS–/– mice (Figure 3).



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Fig. 3. DNA fragmentation in free tumor cells. Mice were inoculated with EATCs, as described in Figure 1. EATCs were obtained from ascites in WT mice 0.5 (lane 1), 1.0 (lane 2), 1.5 (lane 3) and 2 (lane 4) weeks after tumor cell inoculation. Tumor cells were also obtained from iNOS-deficient mice 1.5 (lane 5) and 2 (lane 6) weeks after tumor cell inoculation. DNA samples were extracted from 2 x 106 cells and analyzed by agarose gel electrophoresis, as described in the text. Lane M, molecular weight markers. The experiments were repeated five times, and similar results were obtained.

 
Effects of various agents on cultured EATCs
To obtain further insights into the mechanism by which NO induces death of EATCs, the effect of NO donors on the fate of cultured tumor cells was observed. NOC18 and SNAP markedly decreased the viability of EATCs (Figure 4). Because NO has been reported to inhibit mitochondrial electron transport and ATP synthesis, the effects of rotenone, antimycin A (both mitochondrial electron transport inhibitors) and oligomycin (an ATPase inhibitor) were analyzed. These agents also decreased the viability of EATCs, but to a lesser extent than NO donors.



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Fig. 4. Effect of NO on tumor cells. EATCs (1 x 105/ml) were cultured in the presence of 100 µM NOC18, 100 µM SNAP, 1 µM rotenone, 0.1 µM antimycin A or 50 ng/ml oligomycin. The control bar indicates the cell viability before treatment. Cell viability was estimated by microscopic counting of EATCs that excluded trypan blue. Data are expressed as means ± SD (n = 7). The difference is significant between the control and all groups in the presence of various agents (*P < 0.05).

 
Subcutaneous growth of EATCs in the solid form and levels of NO metabolites
Subcutaneously inoculated EATCs grew rapidly and formed a solid tumor in WT mice, but failed to grow in iNOS–/– mice (Figure 5). These results clearly indicate that NO generated by iNOS predominantly enhances the growth of solid tumors. Plasma levels of NO metabolites increased with time in WT mice, but not in iNOS–/– mice, following subcutaneous inoculation of EATCs (Figure 6).



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Fig. 5. Growth of solid tumor cells. Mice were inoculated subcutaneously with EATCs (106 cells/mouse) to allow growth of a solid tumor. The size of the tumor was assessed in a blinded, coded fashion every 5 days, and recorded by measuring the largest perpendicular diameters with calipers. WT, open circles; iNOS-deficient, closed circles. Data are reported as the average tumor area ± SD (n = 10).

 


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Fig. 6. NO metabolites in plasma from mice subcutaneously inoculated with tumor cells. Mice were inoculated with EATCs, as described in Figure 5. Plasma samples were collected and concentrations of NO metabolites () were determined, as described in the text. WT, open bars; iNOS-deficient, closed bars. Data are expressed as means ± SD (n = 10). *P < 0.05 versus iNOS-deficient at 2 W.

 
Expression of iNOS in solid tumors
To investigate the responses of the animals, iNOS protein was determined with solid tumors. Western blot analysis revealed that iNOS was expressed markedly in WT but not in iNOS–/– (Figure 7A).



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Fig. 7. Expression of iNOS and VEGF mRNA in solid tumor EATCs. Mice were inoculated with EATCs, as described in Figure 5. Tumor lysates (10 µg protein) were subjected to 7.5% SDS–PAGE for western blot analysis (A). Tumor mRNAs for VEGF and ß-actin were analyzed by RT–PCR (B). PCR for VEGF and ß-actin was performed for 30 cycles. The experiments were repeated three times, and similar results were obtained.

 
Expression of VEGF mRNA in solid tumors
Expression of VEGF121 was much higher in WT mice than in iNOS–/– mice, while the difference in VEGF165 expression was not as marked (Figure 7B).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present work shows that the growth of tumor cells is markedly inhibited in subcutaneous tissues of iNOS–/– mice compared with controls, but enhanced in the peritoneal cavity compared with controls. These results clearly indicate the iNOS-derived NO plays pivotal roles in the proliferation of tumor cells, by inhibiting the growth of free cells, but enhancing that of solid tumor cells.

We reported previously that NO reversibly interacts with terminal oxidases in the electron transport chains of mitochondria, thereby inhibiting the respiration of mammalian cells under low oxygen tension (5). It should be noted that inoculation of tumor cells elicits inflammatory reactions that induce infiltration of iNOS-expressing macrophages at the site of inoculation (4,6). Because the oxygen tension in the peritoneal cavity is fairly low (5), energy transduction in the mitochondria of transplanted tumor cells will be inhibited strongly by macrophage-derived NO. Since DNA synthesis and recruitment of tumor cells into S-phase both require large amounts of ATP (10), macrophage-derived NO would principally be expected to suppress the proliferation of tumor cells. It has been well documented that inhibition of the mitochondrial electron transport system increases cytosolic levels of Ca2+ (5), thereby activating Ca2+-dependent processes. Inhibition of the mitochondrial electron transport system induces a membrane permeability transition and releases cytochrome c, a prerequisite to apoptosis (11). Furthermore, activation of endonucleases by Ca2+ also enhances fragmentation of nuclear DNA (12). Thus, it is not surprising that the growth of inoculated EATCs in the peritoneal cavity was inhibited by NO in WT mice, but not in iNOS–/– mice. The findings in the present study that the decrease in the number of peritoneal tumor cells became apparent 2 weeks after inoculation, with a concomitant increase in the generation of NO, is consistent with this hypothesis.

We have also reported previously that no DNA fragmentation was apparent in ascites tumor cells 1 week after inoculation when the rate of NO generation was low, while marked fragmentation of DNA was induced by intraperitoneal administration of NO donors (4). DNA fragmentation of the tumor cells became apparent 2 weeks after inoculation when endogenous generation of NO became significant, while administration of a potent iNOS inhibitor strongly suppressed apoptosis. Furthermore, antimycin A also induced fragmentation of tumor cell DNA by a mechanism that was inhibited by addition of TMPD and ascorbate, which are electron donors for mitochondrial terminal oxidase. These observations indicate that inhibition of mitochondrial electron transport by endogenously generated NO effectively induces apoptosis of ascites tumor cells. Thus, NO plays critical roles in host defense against tumor cells by inhibiting their energy metabolism and/or through an apoptotic mechanism, particularly under low oxygen tension in the peritoneal cavity. In the present study, it should be noted that the effect of NO donors on cell viability was more marked than that of mitochondrial electron transport inhibitors. Because NO has been reported to react with various heme proteins and modulate their functions, NO is also likely to affect systems other than mitochondria.

In contrast to the rapid growth of EATCs in the peritoneal cavity of iNOS–/– mice, tumor cell growth in the subcutaneous tissue was strongly inhibited in iNOS–/– mice, compared with that in WT animals. It has been well documented that the growth of a solid tumor strongly depends on neovascularization and vascular permeability; both factors are important for supplying nutrients to proliferating tumor cells (13,14). Macrophages use L-arginine to synthesize NO through iNOS. The released NO can contribute to the tumoricidal activity and it has been suggested that among the various effector mechanisms considered to be involved in tumor cell killing by activated macrophages, L-arginine-dependent reactive nitrogen intermediates appear to hold a major role (4,15). Interestingly, however, iNOS expression by tumor-associated macrophages has been shown to increase blood flow (16) and angiogenesis (17). Thus, the elevated iNOS expression by tumor-associated macrophages in the stroma of carcinomas, which correlated with advanced tumor grade (18), suggests a balance in favor of vascular effects rather than tumoricidal effects. NO is known to increase both vascular permeability and angiogenesis by up-regulating VEGF (19,20). Hence, VEGF expression enhanced by inflammation might be significantly lower in iNOS–/– mice than in WT animals, and thus insufficient expression of VEGF around tumor cells may explain the strong inhibition of the growth of subcutaneously transplanted EATCs in iNOS–/– mice. In the present study, the expression of VEGF121 was much higher in WT than in iNOS–/– mice. At least six differentially spliced variants of VEGF are known, giving rise to mature isoforms containing 121, 145, 165, 183, 189 and 206 aa. However, little is yet known concerning the in vivo function of this differential splicing, although it has been reported that the VEGF121 isoform is both more angiogenic and tumorigenic than other isoforms (21).

It should be noted that NO levels were higher in plasma than ascites of WT with the growth of free tumor cells. The expression of iNOS was found in the peritoneal macrophages, liver and spleen (data not shown). Thus, the production of NO could depend not only on the peritoneal macrophages, but also on other systemic reticular tissues.

Although the number of EATCs started to decrease 2 weeks after intraperitoneal inoculation, all the WT animals died within 2–3 weeks. On the other hand, despite the rapid and extensive growth of tumor cells in iNOS–/– mice, these animals survived significantly longer (at least 4–5 weeks) than the WT animals. The prolonged survival of iNOS–/– could be the failure of the tumor cells in these animals to implant and form solid tumors or metastasize. Because cachexia is a major cause of death in patients with advanced cancer (22), iNOS-derived NO might enhance the pathological metabolism that in turn enhances cachexia. Hence, apart from its role in proliferation of tumor cells, selective modulation of iNOS activity may have therapeutic potential. We are currently investigating the molecular mechanism through which cachexia is enhanced via NO-dependent metabolism.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received March 26, 2004; revised May 27, 2004; accepted June 10, 2004.





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