- Email: [email protected]
In vitro and in vivo cytotoxic effects of nitric oxide on metastatic cells
CANCER LE’ITERS
i
! Cancer Letters 115 (1997) 57-62
In vitro and in vivo cytotoxic effects of nitric oxide on metastatk cells Seishiro Hirano* Regional Environment Division, National Institute for Environmental Studies, 16-2 Onogarva, Tsukuba. Ibaraki 305, hparr Received 20 September 19996;revision received 5 November 1996; accepted 13 January 1997
Abstract It has been reported that nitric oxide (NO) is tumoricidal in vitro and inhalation of NO is effective for the therapy of pulmonary hypertension. However, little atte$ion has been addressed to the effects of inhaled NO on tumors in the lung. In the present study cytotoxic effects of NO have been investigated both in vitro and in vivo using meta&atic cell lines. Viabilities of both B16 melanoma and Lewis lung carcinoma cells were decreased in the presence of Snitroso-N-acetylDr.-penicillamine (SNAP) in vitro and the cytotoxicity of SNAP was reduced dose-dependently by NO radical scavenger, oxyhemoglobin. To examine in vivo tumoricidal activity of NO, mice were exposed to lo-80 ppm NO gas after intravenous injection of both tumor cell lines. Intravenous injection of both cell lines prodWed metastic tumor coh%&zs in the mouse lung. However, inhaled NO did not reduce the tumor colony formation in the lung. The increase in NO concent&cm was accompaniedby elevation of concomitant nitric dioxide concentration in exposure chambers and @xposum to higher concentration of NO appeared to enhance tumor colony formation in the lung. 0 1997 Elsevier Science Ireland Ltd. Keywords:
Nitric oxide; Inhalation; B16 melanoma; Lewis lung carcinoma; Metastasis
1. Intmduetion It has been reported that murine macrophages activated by lipopolysaccharide or INFr produce nitric oxide (NO) [ 1,2] and NO inhibits mitochondrial respiration 13-51. aconitase activity [5] and DNA synthesis of target cells 15-101. Those cytotoxic effects depend on the supply of L-arginine and are reduced by a competitive nitric oxide synthase (NOS) inhibitor, p-monomethylarginine [3,8-121. The cytotoxic effect of NO on tumor cells has also been demonstrated
by an inverse correlation
* Tel./fax: +8 1 298 5025 12; e-mail: [email protected]
between
inducible NOS activity and production of metetstasis in K-1735 murine melanoma cells [12]. On the other hand, NO has been reported to be a potential car&ogen, because NO suppresses T cell proliferation, damages DNA double strands and forms nitrosoamine H31. It has been proven that endo&&um-derived relaxing factor is identical to NO that is produced by
endotheIial NOS [ 14,151 and i&aL&oa of NO gas is effective for the therapy of pulmonary hypertension [16-191. Up to 80 ppm of NO has been commonly adopted for clinical treatment [l&19]. However, pulmonary toxicity of inhaled NO is controversial. Some investigators reported that NO is protective against reactive oxygen-mediated lung injury 120,211. On the other hand, peroxynitrite, a product of NO and
0304-3835/97/$17.00 0 1997 Elsevier Science Ireland Ltd. All rights reserved PII s0304-3835(97)04706-x
58
S. Hiram / Cancer Letters 115 (1997) 57-62
Male C57BL/6J mice were purchased from Clea Japan (Tokyo) and maintained in an air-conditioned clean room (25”C, relative humidity 50-55%). They were allowed free access to distilled water and commercial chow during the experimental period.
cells/ml and 100 ~1 of the cell suspension was aliquoted into a 96-well culture dish (Costar). After 30 h of culture half of the medium was replaced with fresh medium and S-nitroso-N-acetyl-DL-penicillamine (SNAP, Wako Pure Chem.) and human oxyhemoglobin (Calzyme, San Luis Obispo, CA) were added to final concentrations of 2 mM and O-l mg/ ml, respectively. The stock SNAP solution was prepared in demethylsulfoxide (DMSO) at 400 mM and DMSO alone was added to the control wells to a final concentration of 0.5%. After 24 h of culture in the presence or absence of SNAP or oxyhemoglobin, the monolayer was washed gently with Hanks’ balanced salt solution. The viability of the cells was measured using a modified MTT assay. Briefly, WST1 solution (Wako Pure Chem., dissolved in DMEM without phenol red) was added to each well and the cells were incubated at 37°C for 1.5 h. After the incubation, O.D. at 450 nm was measured with a reference of 650 nm using a microtiter plate reader (CS9300, Shimadzu, Kyoto, Japan).
2.2. Tumor cell lines
2.4. NO gas
Lewis lung carcinoma and B16 melanoma-4A5 were obtained from RIKEN Cell Bank (Tsukuba, Japan) and subcultured in Dulbecco’s modified essential medium (DMEM) containing 10% fetal bovine serum (FBS). Highly metastatic cells were selected in vivo according to Fidler’s method [24]. Briefly, 5 x lo5 viable cells were injected into the tail vein of the mouse and they were killed by severing abdominal aorta under halothane anesthesia 2-3 weeks after the injection. Tumor colonies were taken aseptically and digested into cell suspension by incubating in DMEM containing 0.1% collagenase (Wako Pure Chem., Osaka, Japan) for 90 min. The cells were centrifuged, washed and resuspended in DMEM containing 10% FBS and plated into the culture dish (60 mm+, Costar, MA). The in vivo selection of both cell lines was repeated six times and the selected cell lines were designated as LLC-F6 and B 16-4A5F6, respectively.
NO was supplied from nitrogen-balanced NO gas cylinders (4000 and 20000 ppm) through thermal mass-flow controllers. NO gas was diluted just before the exposure chamber (20 1) with filtered air (25”C, relative humidity 50-55%) to obtain predetermined NO concentrations. The flow rate was set at 5 l/min for each chamber. NO and nitrogen dioxide (NO*) concentrations in the exposure chambers were measured according to the triethanolamine method [25] using NOx (NO/NO*) samplers (A7401, Tokyo Chem. Ind., Tokyo). Briefly, N4, samplers were placed in the inhalation chamber for 48 h before and after the inhalation experiment. The absorbent papers in both NO2 and NO + NO* (containing an oxidizing reagent) compartments were immersed in distilled water to extract nitrite ion and the nitrite concentration was measured calorimetrically using Griess reaction [4]. NO and NO2 concentrations in the exposure chamber are summarized in Table 1.
2.3. Tumoricidal activity of NO in vitro
2.5. Exposure of mice to NO gas
LLC-F6 and B16-4A5-F6 were suspended in DMEM containing 10% FBS at 0.15 x lo6 viable
Groups of seven mice (5 weeks old) were injected with 1 x lo5 viable cells of B16-4A5-F6 into the tail
superoxide or concomitant nitrogen dioxide (NOz) have been shown to cause lung injury [ 19,22,23] Although there are many studies concerning NOmediated lysis of tumor cells in vitro and therapeutics of pulmonary hypertension by inhaled NO, no attempt has been done to investigate inhibitory effects of inhaled NO on tumor colony formation in the lung. The present study was undertaken to study tumoricida1 activity of NO both in vitro and in vivo using metastatic cell lines.
2. Materials and methods 2.1. Animals
59
S. Hirano / Cancer tatters 115 { 1997) 57-G! Table 1 Exposure condition of NO
LLC-F6 (air) LLC-Fh (low NO) LLC-F6 (high NO) B 16-4A5-F6 (air) B 16-4A5-F6 (low NO) B16-4A5-F6 (high NO)
Exposure period (weeks)
Target concentration of NO (ppm)
Concentration of NO in the chamber (ppm)
Concentration NO2 in
2 2 2 3 3 3
0 20 100 0 10 50
N.D. 18.7 80.7 ND. 9.35 49.7
N.D. 1.10 19 I N.D. 0.285 6.98
the chamber
(ppm)
N.D., not detected
vein. After the injection, they were housedin stainless wire cagesand exposed to 0, 10, and 50 ppm of NO gas for 21 days. Other groups of 6-7 mice (6 weeks old) were injected with 1.0 x lo5 viable cells of LLCF6 and exposed 0,20, and 80 ppm of NO gas for 14 days. The exposure was interrupted for 30 min every week for food and water supply. After tie exposure, the mice were killed by severing the abdognal aorta. The lungs were excised, rinsed in Sal&e, blotted, weighed and fixed in Bouin’s solution (Sigma, St. Louis, MO). Tumor colonies were enumeratedusing a dissecting microscope.
3.2. Efects
of inhaled NO
Table 2 shows effects of inhaled NO on tumor colony formation in the lung. Intravenous injection of LLC-F6 and B16-4A5-F6 resulted in 53-194 (mean 88) and 19-87 (mean 41) tumor colony form&on in the lung, respectively. Contrary to my expectation, inhalation exposure of those mice to NO gas did not reduce the number of tumor colonies The lung wet weight of tumor-bearing mice was s&&tly increased compared to that of control mice. Exposure to 80.7
2.6. Statistics
Values are means k SEM. Statistical analysis of the in vivo data was carried out by one-way analysis of variance with Bonferroni’s post hoc comparison and probability value less than 0.05 was acceptedas indicative of significant difference.
$
3. Redts 3.1. In vitro cytotoxicity
” E 33 3
of NO
Fig. 1 shows that SNAP releasedNO in the culture medium almost linearly with time for at least 24 h. Fig. 2A,B shows that viabilities of LLC-F6 and B164AJ-F6 decreasedto 30 and 58% of their control value, respectively, after 24 h of culture in the presence of 2 mM SNAP. The cytotoxic effects of NO were reduced dose-dependentlyby a NO radical scavenger, oxyhemoglobin.
10
15
25
Time (hr) Fig. 1. Release of NO from SNAP in cultnre medium. presented as means of triplicate replicates.
Data are
60
S. Hirano /Cancer Letters 115 (1997) 57-62
Table 2 Effects of NO exposure on lung wet weight and tumor colony formation in the mouse lung
Control LLC-F6 (air) LLC-F6 (low NO) LLC-F6 (high NO) B16-4A5-F6 (air) B16-4A.5-F6 (low NO) B 16-4A5-F6 (high NO) *Significantly
Number of mice
Lung wet weight (g) (mean f SEM)
Number of tumor colonies (mean k SEM)
6 6 I I I 7 7
0.121 0.153 0.160 0.194 0.138 0.136 0.143
Ok0 88 ?r 22 103 * 39 108 f 20 41 zk 8.4 34 k 1.3 37 + 8.3
?r 0.0019 f 0.0062 f 0.0137 + 0.0165* + 0.0036 f 0.0037 f 0.0046
different from the corresponding air-exposed group.
ppm NO (containing 19.1 ppm NO*) for 2 weeks significantly increased lung wet weight in LLC-F6injected mice. 4. Discussion It has been demonstratedthat LPS- or II++stimulated macrophageskill tumor cells and NO is the most effective molecule for the cytotoxic effects [3,8-121. In the present in vitro study both viable LLC-F6 and B16-4A5-F6 cells were decreasedin the presenceof SNAP and the cytotoxic effects of SNAP was reduced by oxyhemoglobin, suggestingthat both cell lines are sensitive to NO. Some circulating metastatic tumor cells, which escapedfrom the immune system,adhereto epithelial cells, migrate into the tissue, proliferate, and make colonies in the lung [24]. Inhaled NO is effective for the therapy of pulmonary hypertension [ 16-191 and chronic obstructive lung diseases [26]. It has been reported that excessNO binds readily to hemoglobin [27], suggesting that alveolar NO diffuses through epithelial, interstitial and endothelial layers. Thus, it is reasonableto postulate that inhaled NO may reduce the viabilities of those tumor cells or visible tumor colonies in the lung as well as in the culture system. However, as shown in Table 2, inhaled NO did not reduce the number of tumor colonies in B16-4A5injected mice, and appeared to enhance the tumor colony production slightly in LLC-F6-injected mice. It should be noted that exposureto high concentration of NOx (80.7 ppm NO and 19.1 ppm NO*) significantly increasedthe lung wet weight in the LLC-F6-
injected group. The increase in the lung wet weight may have been caused by NO&duced lung injury, because it is well known that NO* (0.3420 ppm) causesinterstitial edema and hyperplasia in the lung [28-3 11.It hasbeenreported that exposureto 0.1-0.8 ppm ozone for 1- 14 days resulted in enhancementof pulmonary metastasis in fibrosarcoma (NR-FS)injected mice [32]. Those observations suggest that oxidative stress may enhance pulmonary metastasis of tumor cells. The enhancementof pulmonary metastasis may be causedby suppressionof NK activity and increased possibility of tumor cell lodging in lung microvasculature [32]. It hasbeen shown that NO concentration of tobacco smokeis 4OO-1000ppm with an undetectablelevel of NO2 [33], suggesting that NO is relatively stable in reducing flame or atmosphereand up to 0.1% NO is not lethally toxic in the absenceof NO*. However, as shown in the present study, about 19 ppm NO2 was concomitant with about 81 ppm NO even in the small and well-ventilated chambers.Using NO2 absorbents such as sodalime and triethanolamine at the inlet did not reduce NO2 concentration significantly in the chamber (data not shown), suggesting that NO-NO2 equilibrium is reached rapidly in the air. Thus, it seemsto be difficult to perform common inhalation exposuresof laboratory small animals to high concentration of pure NO. In summary, although both B 16-4A5-F6 and LLCF6 are sensitive to NO in vitro, inhalation exposureof tumor-bearing mice to NO gas (up to 80 ppm) appearedto be ineffective for those two cell lines in vivo. A significant amount of NO2 was generatedin the exposure chamber, when NO concentration was
S. Hirano / Cancer I&ten
I15 (1997) 57 62
hl
References
100
-
(A)
0 SNAP (2 mM) Oxy-Hb (mg/ml)
0
+
+
+
+
0
0.05
0.2
1
03
_
0 SNAP (2 mM Oxy-Hb (mg/ml)
0
+
+
+
+
0
0.05
0.2
1
Fig. 2. Cytotoxic effects of SNAP on LLC-F6 (A) and B16-4A5-F6 cells (B) and reduction of the cytotoxic effects by oxyhemoglobin. The viability of the cells in the presence of 2 mM SNAP is shown as percent of control value (hatched column).
increased. It remains to be answered whether exposure to a higher concentration of NO (for example 0.1%) with much less NO2 decreases tumor colony production in the lung.
Acknowledgements The author thanks Mrs. R. Liu for her skillful assistance in the experiments.
[I] Stnehr, D.J. and Marietta, M.A. (1985) Mammalian nitrate biosynthesis: mouse macrophages produce nitrite and nitrate in response to Escherichia coli lipopolysaccharide, Proc. Natl. Acad. Sci. USA. 82, 7738-7742. 121 Lavnikova, N., Drapier, J.-C. and Laskin, D.1. (1993) A single exogenous stimulus activates resident rat macrophages for nitric oxide production and tumor cytotoxicity, J. Leukoc. Biol., 54, 322,-328. 131 Hibbs, J.B. Jr., Vavrin, 2. and Taintor. R.R. (1987) r.--Arginine is required for expression of the activated macrophage effector mechanism causing selective metabolic inhibition in target cells, J. Immunol., 138, 550-565. [4] Stuehr, D.J. and Nathan, C.F. (1989) A macmphage product responsible for cytostasis and respiratory inhibition in tumor target cells. J. Exp. Med., 169, 1543-1555. [S] Hibbs, J.B. Jr., Taintor, R.R., Vavrin, Z. and Rachlin, E.M. (1988) Nitric oxide: a cytotoxic activated macrophage effector molecule, Biochem. Biophys. Res. Commun., 157, X7-94. [6] Kwon, N.S., Stuehr, D.J. and Nathan, CF. (1991) Inhibition of tumor cell ribonucleotide reductase by macrophagederived nitric oxide, J. Exp. Med., 174, 761. 767. [7] Lepoivre, M., Fhunan, J.-M., Bob& P.. Lemaire, Ci. and Henry, Y. (1994) Quenching of the tyrosyl free radicltl of ribonucleotide reductase by nitric oxide. .I Biol. C’hem.. 269, 21891-21897. [S] Lepoivre. M., Chenais, B., Yapo, A., Lemaire. G.. Thelander. L. and Tenu, .1.-P.(1990) Alterations of ribonucleotide reductase activity following induction of the nitritegenerating pathway in adenocarcinoma cells. J. Rio1 Cbem., 26.5. 14143-14149. [9] Lepoivre. M., Fieschi, F., Coves, J., Thelander, 1.. and Fontecave, M. ( 1991) Inactivation of ribonucleottde reductase by nitric oxide, B&hem. Biophys. Res. Connnun.. 179.342.. 448. [ IO] Keller-, R., Geiges, IM. and Keist, R. ( 29YO)I.-.>nine-dependent reactive nitrogen intermediates as mediators of tumor cell killing hy activated macrophages. Cancer Rec.. 50. 1421--1425. [ 11] Hirano. S. ( 1996) Interaction of alveolar rnacrophages with exposure pulmonary epithelial cells following to lipopolysaccharide, Arch. Toxicol., 70, ‘230 2% [ 121 Dong, Z., Staroselsky, A.H., Qi, X. and Fidler, I.J. I 1994) Inverse correlation between expression ot mducible nitric oxide synthase activity and production of metastasis jn K1735 murine melanoma cells, Cancer Res.. 54, 789-793. [13] Ohshima, H. and Bartsch, H. (1994) Chronic infectious and inflammatory processed as cancer risk factors: possible role of nitric oxide in carcinogenesis, Mutat. Res., 305. 253264. [14] Lowenstein. C.J. and Snyder, S.H. (1992: Nitric oxide: a novel biologi,: messenger, Cell, 70, 705-707 [IS] Zapol, W.M., Rimar, S., Gillis, N.. Marietta. %I. and Rosken, C.H. (1994) .Nitric oxide and the lung. Am. .I. Respir. Crit. Care Med.. 149, 1375-1380. [16] Pepke-Zaba, J., Higenbottam, T.W., Dinh-Xulm. A.T., Stone. D. and Wallwork, J. (1991) Inhaled nitric okrde as a cause of
62
S. Hiram /Cancer Letters 115 (1997) 57-62
selective pulmonary vasodilation in pulmonary hypertension, Lancet, 338, 1173-l 174. [ 171 Gustafsson, L.E. (1993) Experimental studies on nitric oxide, Stand. J. Work Environ. Health, 19 (Suppl. 2), 44-49. [18] Adatia, I., MRCP (UK), FRCP (C) and Wessel, D.L. (1994) Therapeutic use of inhaled nitric oxide. Curr. Opin. Pediatr., 4, 583-590. [19] Miller, O.I., Celermajer, D.S., Deanfield, J.E. and Macrae, D.J. (1994) Guidelines for the safe administration of inhaled nitric oxide, Arch. Dis. Child., 70, F47-F49. [20] Guidot, D.M., Repine, M.J., Hybertson, B.M. and Repine, J.E. (1995) Inhaled nitric oxide prevents neutrophil-mediated, oxygen radical-dependent leak in isolated rat lungs, Am. J. Physiol., 269, L2-L5. [21] Wink, D.A., Hanbauer, I., Laval, F., Cook, J.A., Krishna, M.C. and Mitchell, J.B. (1994) Nitric oxide protects against the cytotoxic effects of reactive oxygen species, Ann. N. Y. Acad. Sci., 738, 265-278. [22] Radi, R., Beckman, J.S., Bush, K.M. and Freeman, B.A. (1991) Peroxynitrite oxidation of sullhydryls, J. Biol. Chem., 266, 4244-4250. [23] Freeman, B. Free radical chemistry of nitric oxides: looking at the dark side. Chest, 105, 79S-84s. [24] Fidler, I.J. (1978) General considerations for studies of experimental cancer metastasis. In: Methods in Cancer Research, pp. 399-439. Editor: H. Busch. Academic Press, New York.
[25] Palmes, E.D. and Tomczyk, C. (1979) Personal samplers for NOx, Am. Ind. Hyg. Assoc. J., 40, 588-591. [26] Adatia, I., Thompson, J., Landzberg, M. and Wessel, D.L. (1993) Inhaled nitric oxide in chronic obstructive lung disease, Lancet, 341, 307-308. [27] Oda, H., Kusumoto, S. and Nakajima, T. (1975) Nitrosylhemoglobin formation in the blood of animals exposed to nitric oxide, Arch. Environ. Health, 30, 453-455. [28] von Nieding, G. and Wagner, H.M. (1979) Effects of NO2 on chronic bronchitics, Environ. Health Perspect., 29, 137142. [29] Sherwin, R.P. and Richters, V. (1982) Hypereplasia of Type 2 pneumocytes following 0.34 ppm nitrogen dioxide exposure: quantitation by image analysis, Arch. Environ. Health, 37, 306315. [30] Hayashi, Y ., Kohno, T. and Ohwada, H. (1987) Morphological effects of nitrogen dioxide on the rat lung, Environ. Health Perspect., 73, 135-145. [31] Gaston, B., Drazen, J.M., Loscalzo, J. and Stamler, J.S. (1994) The biology of nitrogen oxides in the airways, Am. J. Respir. Crit. Care Med., 149, 538-551. [32] Kobayashi, T., Todoroki, T. and Sato, H. (1987) Enhancement of pulmonary metastasis of murine fibrosarcoma NR-FS by ozone exposure, J. Toxicol. Environ. Health, 20, 135145. [33] Norman, V. and Keith, C.H. (1965) Nitrogen oxides in tobacco smoke, Nature, 205, 915-916.
i
! Cancer Letters 115 (1997) 57-62
In vitro and in vivo cytotoxic effects of nitric oxide on metastatk cells Seishiro Hirano* Regional Environment Division, National Institute for Environmental Studies, 16-2 Onogarva, Tsukuba. Ibaraki 305, hparr Received 20 September 19996;revision received 5 November 1996; accepted 13 January 1997
Abstract It has been reported that nitric oxide (NO) is tumoricidal in vitro and inhalation of NO is effective for the therapy of pulmonary hypertension. However, little atte$ion has been addressed to the effects of inhaled NO on tumors in the lung. In the present study cytotoxic effects of NO have been investigated both in vitro and in vivo using meta&atic cell lines. Viabilities of both B16 melanoma and Lewis lung carcinoma cells were decreased in the presence of Snitroso-N-acetylDr.-penicillamine (SNAP) in vitro and the cytotoxicity of SNAP was reduced dose-dependently by NO radical scavenger, oxyhemoglobin. To examine in vivo tumoricidal activity of NO, mice were exposed to lo-80 ppm NO gas after intravenous injection of both tumor cell lines. Intravenous injection of both cell lines prodWed metastic tumor coh%&zs in the mouse lung. However, inhaled NO did not reduce the tumor colony formation in the lung. The increase in NO concent&cm was accompaniedby elevation of concomitant nitric dioxide concentration in exposure chambers and @xposum to higher concentration of NO appeared to enhance tumor colony formation in the lung. 0 1997 Elsevier Science Ireland Ltd. Keywords:
Nitric oxide; Inhalation; B16 melanoma; Lewis lung carcinoma; Metastasis
1. Intmduetion It has been reported that murine macrophages activated by lipopolysaccharide or INFr produce nitric oxide (NO) [ 1,2] and NO inhibits mitochondrial respiration 13-51. aconitase activity [5] and DNA synthesis of target cells 15-101. Those cytotoxic effects depend on the supply of L-arginine and are reduced by a competitive nitric oxide synthase (NOS) inhibitor, p-monomethylarginine [3,8-121. The cytotoxic effect of NO on tumor cells has also been demonstrated
by an inverse correlation
* Tel./fax: +8 1 298 5025 12; e-mail: [email protected]
between
inducible NOS activity and production of metetstasis in K-1735 murine melanoma cells [12]. On the other hand, NO has been reported to be a potential car&ogen, because NO suppresses T cell proliferation, damages DNA double strands and forms nitrosoamine H31. It has been proven that endo&&um-derived relaxing factor is identical to NO that is produced by
endotheIial NOS [ 14,151 and i&aL&oa of NO gas is effective for the therapy of pulmonary hypertension [16-191. Up to 80 ppm of NO has been commonly adopted for clinical treatment [l&19]. However, pulmonary toxicity of inhaled NO is controversial. Some investigators reported that NO is protective against reactive oxygen-mediated lung injury 120,211. On the other hand, peroxynitrite, a product of NO and
0304-3835/97/$17.00 0 1997 Elsevier Science Ireland Ltd. All rights reserved PII s0304-3835(97)04706-x
58
S. Hiram / Cancer Letters 115 (1997) 57-62
Male C57BL/6J mice were purchased from Clea Japan (Tokyo) and maintained in an air-conditioned clean room (25”C, relative humidity 50-55%). They were allowed free access to distilled water and commercial chow during the experimental period.
cells/ml and 100 ~1 of the cell suspension was aliquoted into a 96-well culture dish (Costar). After 30 h of culture half of the medium was replaced with fresh medium and S-nitroso-N-acetyl-DL-penicillamine (SNAP, Wako Pure Chem.) and human oxyhemoglobin (Calzyme, San Luis Obispo, CA) were added to final concentrations of 2 mM and O-l mg/ ml, respectively. The stock SNAP solution was prepared in demethylsulfoxide (DMSO) at 400 mM and DMSO alone was added to the control wells to a final concentration of 0.5%. After 24 h of culture in the presence or absence of SNAP or oxyhemoglobin, the monolayer was washed gently with Hanks’ balanced salt solution. The viability of the cells was measured using a modified MTT assay. Briefly, WST1 solution (Wako Pure Chem., dissolved in DMEM without phenol red) was added to each well and the cells were incubated at 37°C for 1.5 h. After the incubation, O.D. at 450 nm was measured with a reference of 650 nm using a microtiter plate reader (CS9300, Shimadzu, Kyoto, Japan).
2.2. Tumor cell lines
2.4. NO gas
Lewis lung carcinoma and B16 melanoma-4A5 were obtained from RIKEN Cell Bank (Tsukuba, Japan) and subcultured in Dulbecco’s modified essential medium (DMEM) containing 10% fetal bovine serum (FBS). Highly metastatic cells were selected in vivo according to Fidler’s method [24]. Briefly, 5 x lo5 viable cells were injected into the tail vein of the mouse and they were killed by severing abdominal aorta under halothane anesthesia 2-3 weeks after the injection. Tumor colonies were taken aseptically and digested into cell suspension by incubating in DMEM containing 0.1% collagenase (Wako Pure Chem., Osaka, Japan) for 90 min. The cells were centrifuged, washed and resuspended in DMEM containing 10% FBS and plated into the culture dish (60 mm+, Costar, MA). The in vivo selection of both cell lines was repeated six times and the selected cell lines were designated as LLC-F6 and B 16-4A5F6, respectively.
NO was supplied from nitrogen-balanced NO gas cylinders (4000 and 20000 ppm) through thermal mass-flow controllers. NO gas was diluted just before the exposure chamber (20 1) with filtered air (25”C, relative humidity 50-55%) to obtain predetermined NO concentrations. The flow rate was set at 5 l/min for each chamber. NO and nitrogen dioxide (NO*) concentrations in the exposure chambers were measured according to the triethanolamine method [25] using NOx (NO/NO*) samplers (A7401, Tokyo Chem. Ind., Tokyo). Briefly, N4, samplers were placed in the inhalation chamber for 48 h before and after the inhalation experiment. The absorbent papers in both NO2 and NO + NO* (containing an oxidizing reagent) compartments were immersed in distilled water to extract nitrite ion and the nitrite concentration was measured calorimetrically using Griess reaction [4]. NO and NO2 concentrations in the exposure chamber are summarized in Table 1.
2.3. Tumoricidal activity of NO in vitro
2.5. Exposure of mice to NO gas
LLC-F6 and B16-4A5-F6 were suspended in DMEM containing 10% FBS at 0.15 x lo6 viable
Groups of seven mice (5 weeks old) were injected with 1 x lo5 viable cells of B16-4A5-F6 into the tail
superoxide or concomitant nitrogen dioxide (NOz) have been shown to cause lung injury [ 19,22,23] Although there are many studies concerning NOmediated lysis of tumor cells in vitro and therapeutics of pulmonary hypertension by inhaled NO, no attempt has been done to investigate inhibitory effects of inhaled NO on tumor colony formation in the lung. The present study was undertaken to study tumoricida1 activity of NO both in vitro and in vivo using metastatic cell lines.
2. Materials and methods 2.1. Animals
59
S. Hirano / Cancer tatters 115 { 1997) 57-G! Table 1 Exposure condition of NO
LLC-F6 (air) LLC-Fh (low NO) LLC-F6 (high NO) B 16-4A5-F6 (air) B 16-4A5-F6 (low NO) B16-4A5-F6 (high NO)
Exposure period (weeks)
Target concentration of NO (ppm)
Concentration of NO in the chamber (ppm)
Concentration NO2 in
2 2 2 3 3 3
0 20 100 0 10 50
N.D. 18.7 80.7 ND. 9.35 49.7
N.D. 1.10 19 I N.D. 0.285 6.98
the chamber
(ppm)
N.D., not detected
vein. After the injection, they were housedin stainless wire cagesand exposed to 0, 10, and 50 ppm of NO gas for 21 days. Other groups of 6-7 mice (6 weeks old) were injected with 1.0 x lo5 viable cells of LLCF6 and exposed 0,20, and 80 ppm of NO gas for 14 days. The exposure was interrupted for 30 min every week for food and water supply. After tie exposure, the mice were killed by severing the abdognal aorta. The lungs were excised, rinsed in Sal&e, blotted, weighed and fixed in Bouin’s solution (Sigma, St. Louis, MO). Tumor colonies were enumeratedusing a dissecting microscope.
3.2. Efects
of inhaled NO
Table 2 shows effects of inhaled NO on tumor colony formation in the lung. Intravenous injection of LLC-F6 and B16-4A5-F6 resulted in 53-194 (mean 88) and 19-87 (mean 41) tumor colony form&on in the lung, respectively. Contrary to my expectation, inhalation exposure of those mice to NO gas did not reduce the number of tumor colonies The lung wet weight of tumor-bearing mice was s&&tly increased compared to that of control mice. Exposure to 80.7
2.6. Statistics
Values are means k SEM. Statistical analysis of the in vivo data was carried out by one-way analysis of variance with Bonferroni’s post hoc comparison and probability value less than 0.05 was acceptedas indicative of significant difference.
$
3. Redts 3.1. In vitro cytotoxicity
” E 33 3
of NO
Fig. 1 shows that SNAP releasedNO in the culture medium almost linearly with time for at least 24 h. Fig. 2A,B shows that viabilities of LLC-F6 and B164AJ-F6 decreasedto 30 and 58% of their control value, respectively, after 24 h of culture in the presence of 2 mM SNAP. The cytotoxic effects of NO were reduced dose-dependentlyby a NO radical scavenger, oxyhemoglobin.
10
15
25
Time (hr) Fig. 1. Release of NO from SNAP in cultnre medium. presented as means of triplicate replicates.
Data are
60
S. Hirano /Cancer Letters 115 (1997) 57-62
Table 2 Effects of NO exposure on lung wet weight and tumor colony formation in the mouse lung
Control LLC-F6 (air) LLC-F6 (low NO) LLC-F6 (high NO) B16-4A5-F6 (air) B16-4A.5-F6 (low NO) B 16-4A5-F6 (high NO) *Significantly
Number of mice
Lung wet weight (g) (mean f SEM)
Number of tumor colonies (mean k SEM)
6 6 I I I 7 7
0.121 0.153 0.160 0.194 0.138 0.136 0.143
Ok0 88 ?r 22 103 * 39 108 f 20 41 zk 8.4 34 k 1.3 37 + 8.3
?r 0.0019 f 0.0062 f 0.0137 + 0.0165* + 0.0036 f 0.0037 f 0.0046
different from the corresponding air-exposed group.
ppm NO (containing 19.1 ppm NO*) for 2 weeks significantly increased lung wet weight in LLC-F6injected mice. 4. Discussion It has been demonstratedthat LPS- or II++stimulated macrophageskill tumor cells and NO is the most effective molecule for the cytotoxic effects [3,8-121. In the present in vitro study both viable LLC-F6 and B16-4A5-F6 cells were decreasedin the presenceof SNAP and the cytotoxic effects of SNAP was reduced by oxyhemoglobin, suggestingthat both cell lines are sensitive to NO. Some circulating metastatic tumor cells, which escapedfrom the immune system,adhereto epithelial cells, migrate into the tissue, proliferate, and make colonies in the lung [24]. Inhaled NO is effective for the therapy of pulmonary hypertension [ 16-191 and chronic obstructive lung diseases [26]. It has been reported that excessNO binds readily to hemoglobin [27], suggesting that alveolar NO diffuses through epithelial, interstitial and endothelial layers. Thus, it is reasonableto postulate that inhaled NO may reduce the viabilities of those tumor cells or visible tumor colonies in the lung as well as in the culture system. However, as shown in Table 2, inhaled NO did not reduce the number of tumor colonies in B16-4A5injected mice, and appeared to enhance the tumor colony production slightly in LLC-F6-injected mice. It should be noted that exposureto high concentration of NOx (80.7 ppm NO and 19.1 ppm NO*) significantly increasedthe lung wet weight in the LLC-F6-
injected group. The increase in the lung wet weight may have been caused by NO&duced lung injury, because it is well known that NO* (0.3420 ppm) causesinterstitial edema and hyperplasia in the lung [28-3 11.It hasbeenreported that exposureto 0.1-0.8 ppm ozone for 1- 14 days resulted in enhancementof pulmonary metastasis in fibrosarcoma (NR-FS)injected mice [32]. Those observations suggest that oxidative stress may enhance pulmonary metastasis of tumor cells. The enhancementof pulmonary metastasis may be causedby suppressionof NK activity and increased possibility of tumor cell lodging in lung microvasculature [32]. It hasbeen shown that NO concentration of tobacco smokeis 4OO-1000ppm with an undetectablelevel of NO2 [33], suggesting that NO is relatively stable in reducing flame or atmosphereand up to 0.1% NO is not lethally toxic in the absenceof NO*. However, as shown in the present study, about 19 ppm NO2 was concomitant with about 81 ppm NO even in the small and well-ventilated chambers.Using NO2 absorbents such as sodalime and triethanolamine at the inlet did not reduce NO2 concentration significantly in the chamber (data not shown), suggesting that NO-NO2 equilibrium is reached rapidly in the air. Thus, it seemsto be difficult to perform common inhalation exposuresof laboratory small animals to high concentration of pure NO. In summary, although both B 16-4A5-F6 and LLCF6 are sensitive to NO in vitro, inhalation exposureof tumor-bearing mice to NO gas (up to 80 ppm) appearedto be ineffective for those two cell lines in vivo. A significant amount of NO2 was generatedin the exposure chamber, when NO concentration was
S. Hirano / Cancer I&ten
I15 (1997) 57 62
hl
References
100
-
(A)
0 SNAP (2 mM) Oxy-Hb (mg/ml)
0
+
+
+
+
0
0.05
0.2
1
03
_
0 SNAP (2 mM Oxy-Hb (mg/ml)
0
+
+
+
+
0
0.05
0.2
1
Fig. 2. Cytotoxic effects of SNAP on LLC-F6 (A) and B16-4A5-F6 cells (B) and reduction of the cytotoxic effects by oxyhemoglobin. The viability of the cells in the presence of 2 mM SNAP is shown as percent of control value (hatched column).
increased. It remains to be answered whether exposure to a higher concentration of NO (for example 0.1%) with much less NO2 decreases tumor colony production in the lung.
Acknowledgements The author thanks Mrs. R. Liu for her skillful assistance in the experiments.
[I] Stnehr, D.J. and Marietta, M.A. (1985) Mammalian nitrate biosynthesis: mouse macrophages produce nitrite and nitrate in response to Escherichia coli lipopolysaccharide, Proc. Natl. Acad. Sci. USA. 82, 7738-7742. 121 Lavnikova, N., Drapier, J.-C. and Laskin, D.1. (1993) A single exogenous stimulus activates resident rat macrophages for nitric oxide production and tumor cytotoxicity, J. Leukoc. Biol., 54, 322,-328. 131 Hibbs, J.B. Jr., Vavrin, 2. and Taintor. R.R. (1987) r.--Arginine is required for expression of the activated macrophage effector mechanism causing selective metabolic inhibition in target cells, J. Immunol., 138, 550-565. [4] Stuehr, D.J. and Nathan, C.F. (1989) A macmphage product responsible for cytostasis and respiratory inhibition in tumor target cells. J. Exp. Med., 169, 1543-1555. [S] Hibbs, J.B. Jr., Taintor, R.R., Vavrin, Z. and Rachlin, E.M. (1988) Nitric oxide: a cytotoxic activated macrophage effector molecule, Biochem. Biophys. Res. Commun., 157, X7-94. [6] Kwon, N.S., Stuehr, D.J. and Nathan, CF. (1991) Inhibition of tumor cell ribonucleotide reductase by macrophagederived nitric oxide, J. Exp. Med., 174, 761. 767. [7] Lepoivre, M., Fhunan, J.-M., Bob& P.. Lemaire, Ci. and Henry, Y. (1994) Quenching of the tyrosyl free radicltl of ribonucleotide reductase by nitric oxide. .I Biol. C’hem.. 269, 21891-21897. [S] Lepoivre. M., Chenais, B., Yapo, A., Lemaire. G.. Thelander. L. and Tenu, .1.-P.(1990) Alterations of ribonucleotide reductase activity following induction of the nitritegenerating pathway in adenocarcinoma cells. J. Rio1 Cbem., 26.5. 14143-14149. [9] Lepoivre. M., Fieschi, F., Coves, J., Thelander, 1.. and Fontecave, M. ( 1991) Inactivation of ribonucleottde reductase by nitric oxide, B&hem. Biophys. Res. Connnun.. 179.342.. 448. [ IO] Keller-, R., Geiges, IM. and Keist, R. ( 29YO)I.-.>nine-dependent reactive nitrogen intermediates as mediators of tumor cell killing hy activated macrophages. Cancer Rec.. 50. 1421--1425. [ 11] Hirano. S. ( 1996) Interaction of alveolar rnacrophages with exposure pulmonary epithelial cells following to lipopolysaccharide, Arch. Toxicol., 70, ‘230 2% [ 121 Dong, Z., Staroselsky, A.H., Qi, X. and Fidler, I.J. I 1994) Inverse correlation between expression ot mducible nitric oxide synthase activity and production of metastasis jn K1735 murine melanoma cells, Cancer Res.. 54, 789-793. [13] Ohshima, H. and Bartsch, H. (1994) Chronic infectious and inflammatory processed as cancer risk factors: possible role of nitric oxide in carcinogenesis, Mutat. Res., 305. 253264. [14] Lowenstein. C.J. and Snyder, S.H. (1992: Nitric oxide: a novel biologi,: messenger, Cell, 70, 705-707 [IS] Zapol, W.M., Rimar, S., Gillis, N.. Marietta. %I. and Rosken, C.H. (1994) .Nitric oxide and the lung. Am. .I. Respir. Crit. Care Med.. 149, 1375-1380. [16] Pepke-Zaba, J., Higenbottam, T.W., Dinh-Xulm. A.T., Stone. D. and Wallwork, J. (1991) Inhaled nitric okrde as a cause of
62
S. Hiram /Cancer Letters 115 (1997) 57-62
selective pulmonary vasodilation in pulmonary hypertension, Lancet, 338, 1173-l 174. [ 171 Gustafsson, L.E. (1993) Experimental studies on nitric oxide, Stand. J. Work Environ. Health, 19 (Suppl. 2), 44-49. [18] Adatia, I., MRCP (UK), FRCP (C) and Wessel, D.L. (1994) Therapeutic use of inhaled nitric oxide. Curr. Opin. Pediatr., 4, 583-590. [19] Miller, O.I., Celermajer, D.S., Deanfield, J.E. and Macrae, D.J. (1994) Guidelines for the safe administration of inhaled nitric oxide, Arch. Dis. Child., 70, F47-F49. [20] Guidot, D.M., Repine, M.J., Hybertson, B.M. and Repine, J.E. (1995) Inhaled nitric oxide prevents neutrophil-mediated, oxygen radical-dependent leak in isolated rat lungs, Am. J. Physiol., 269, L2-L5. [21] Wink, D.A., Hanbauer, I., Laval, F., Cook, J.A., Krishna, M.C. and Mitchell, J.B. (1994) Nitric oxide protects against the cytotoxic effects of reactive oxygen species, Ann. N. Y. Acad. Sci., 738, 265-278. [22] Radi, R., Beckman, J.S., Bush, K.M. and Freeman, B.A. (1991) Peroxynitrite oxidation of sullhydryls, J. Biol. Chem., 266, 4244-4250. [23] Freeman, B. Free radical chemistry of nitric oxides: looking at the dark side. Chest, 105, 79S-84s. [24] Fidler, I.J. (1978) General considerations for studies of experimental cancer metastasis. In: Methods in Cancer Research, pp. 399-439. Editor: H. Busch. Academic Press, New York.
[25] Palmes, E.D. and Tomczyk, C. (1979) Personal samplers for NOx, Am. Ind. Hyg. Assoc. J., 40, 588-591. [26] Adatia, I., Thompson, J., Landzberg, M. and Wessel, D.L. (1993) Inhaled nitric oxide in chronic obstructive lung disease, Lancet, 341, 307-308. [27] Oda, H., Kusumoto, S. and Nakajima, T. (1975) Nitrosylhemoglobin formation in the blood of animals exposed to nitric oxide, Arch. Environ. Health, 30, 453-455. [28] von Nieding, G. and Wagner, H.M. (1979) Effects of NO2 on chronic bronchitics, Environ. Health Perspect., 29, 137142. [29] Sherwin, R.P. and Richters, V. (1982) Hypereplasia of Type 2 pneumocytes following 0.34 ppm nitrogen dioxide exposure: quantitation by image analysis, Arch. Environ. Health, 37, 306315. [30] Hayashi, Y ., Kohno, T. and Ohwada, H. (1987) Morphological effects of nitrogen dioxide on the rat lung, Environ. Health Perspect., 73, 135-145. [31] Gaston, B., Drazen, J.M., Loscalzo, J. and Stamler, J.S. (1994) The biology of nitrogen oxides in the airways, Am. J. Respir. Crit. Care Med., 149, 538-551. [32] Kobayashi, T., Todoroki, T. and Sato, H. (1987) Enhancement of pulmonary metastasis of murine fibrosarcoma NR-FS by ozone exposure, J. Toxicol. Environ. Health, 20, 135145. [33] Norman, V. and Keith, C.H. (1965) Nitrogen oxides in tobacco smoke, Nature, 205, 915-916.