Clinical & Experimental Metastasis 20: 445–450, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.
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Different effects of constitutive nitric oxide synthase and heme oxygenase on pulmonary or liver metastasis of colon cancer in mice Takeshi Ishikawa1, Norimasa Yoshida2, Hiroshi Higashihara1 , Mamoru Inoue1 , Kazuhiko Uchiyama1 , Tomohisa Takagi1 , Osamu Handa1 , Satoshi Kokura2 , Yuji Naito2 , Takeshi Okanoue2 & Toshikazu Yoshikawa1 1 Inflammation and Immunology, 2 Molecular Gastroenterology and Hepatology, Graduate School of Medical Science, Kyoto
Prefectural University of Medicine, Kamigyo-ku, Kyoto, Japan Received 29 July 2002; accepted in revised form 21 January 2003
Key words: cancer, carbon monoxide, colon cancer, heme oxygenase, metastasis, nitric oxide, nitric oxide synthase
Abstract It has recently been reported that not only endogenous nitric oxide (NO) but also carbon monoxide (CO) produced by heme oxygenase (HO) have many physiological functions. The objective of the present study was to determine whether endogenous NO or CO is involved in the experimental pulmonary or liver metastasis of colon cancer in mice. Intravenous or intrasplenic injection of colon 26 cells from a mouse colon adenocarcinoma cell line resulted in multiple pulmonary or liver metastases. NG-nitro- L-arginine methyl ester (L-NAME), a competitive inhibitor of NO synthase (NOS), or zinc deuteroporphyrin 2, 4-bis glycol (ZnDPBG), a competitive inhibitor of HO, was administered to the mice only on the day of tumor inoculation. We assessed the number of tumor cells 24 h later and the outcome of metastases of the target organ. In the pulmonary metastasis model, L-NAME increased both the number of tumor cells 24 h later and outcome of metastases 18 days later, but did not have a significant effect on liver metastasis. On the other hand, metastasis to the liver, but not that to the lung, increased following administration of ZnDPBG. These results suggest that the activities of NOS and HO could influence experimental metastasis in an organ-specific manner. Abbreviations: ADP – adenosine diphosphate; CO – carbon monoxide; D-NAME – NG-nitro-D-arginine methyl ester; HO – heme oxygenase; L-NAME – NG-nitro-L-arginine methyl ester; LPS – lipopolysaccharide; NO – nitric oxide; NOS – nitric oxide synthase; PPP – platelet-poor plasma; PRP – platelet-rich plasma; ZnDPBG – L-arginine, zinc deuteroporphyrin 2, 4-bis glycol Introduction Cancer metastasis is a complex, multistep process that involves cell separation from the primary tumor, entry into the vascular system, transport to and arrest within the microcirculation of distant organs and extravasation [1–3]. Nitric oxide (NO) is a multifunctional molecule produced by NO synthase (NOS) from L-arginine [4, 5]. NOS has been reported to be expressed in many cells, such as endothelial cells [6], macrophages [7], vascular smooth muscle cells [8], platelets [9] and several tumor cells [10–12]. NO regulates vasodilatation [13, 14] and inhibits platelet aggregation [9, 15] and platelet adhesion to endothelial cells [16]. Some researchers have reported that NO reduces the expression of endothelial adhesion molecules [17]. NO may be involved in multiple steps that could influence the outcome of metastasis. Previous studies showed an Correspondence to: Norimasa Yoshida, MD, PhD, Molecular Gastroenterology and Hepatology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan. Tel: +81-75-2515508; Fax: +81-75-2523721; E-mail:
[email protected]
inverse correlation between the expression of inducible NOS activity in murine melanoma cells and their metastatic potential [18]. NO might have been reported to affect tumor cell metastasis by interfering with the adhesive process between tumor cells and endothelial cells [19], but Edwards et al. showed that tumor cell NO stimulates experimental metastasis in a murine model [20]. The effect of NO, especially that produced by endothelial NOS, on cancer metastasis remains controversial. Carbon monoxide (CO) produced by heme oxygenase (HO) reaction contributes to the regulation of cell functions by activating soluble guanylate cyclase [21, 22], and thereby CO shares several biological actions with NO, such as smooth muscle relaxation [23] or inhibition of platelet aggregation [21]. Liver is, like the brain and spleen, one of the most abundant sources of HO among organs [21]. Suematsu et al have shown that CO can function as an endogenous modulator of sinusoidal tone in liver [24]. Based on these findings, we hypothesized that endogenous CO could also influence cancer metastasis, especially to the liver.
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Many studies examined the effects of endogenous NO generated from tumor cells on tumor progression and metastasis [12, 18, 25], but there were few studies about the effects of NO generated from host cells, such as endothelial cells and macrophages, and there were few reports regarding the effects of CO on tumor metastasis. In the present study, we investigated the effects of endogenous NO and CO produced by host cells on the arrest of tumor cells in the venous or capillary bed in experimental pulmonary or liver metastasis models using NOS or HO inhibitors.
injected intraperitoneally into mice 3 h and 1 h before and 1 h and 3 h after tumor inoculation. ZnDPBG was dissolved in 4.0 µmol/l Na2 CO3 solution in a volume of 0.14 ml and was injected intraperitoneally into mice 1 h before tumor inoculation. The lungs were removed 18 days after tumor inoculation, and tumor nodules were counted. The liver was removed 11 days after tumor inoculation and liver weight was measured as an index of metastasis.
Materials and methods
For analysis of the retention of tumor cells after tumor inoculation, these cells were labeled with a fluorescent reagent, PKH26, according to the manufacturer’s instructions. After being washed with phosphate-buffered saline, PKH26labeled cells were used for the experiment. We inoculated mice with 2 × 105 PKH26-labeled colon26 cells through a tail vein for the pulmonary experiment or 2 × 106 cells through the spleen for the liver experiment. Drugs were administered as described above. Mice were sacrificed at 24 h after tumor inoculation, and lung or liver fragments were harvested and embedded, frozen in O.C.T. compound with dry ice and cut into 8-µm sections on a cryostat. The sections were dried and examined using a fluorescent microscope.
Animals Male BALB/c mice were purchased from SHIMIZU Laboratory Supplies (Kyoto, Japan). They were fed a standard laboratory diet and water ad libitum under standard laboratory conditions, and used for experiments at the age of eight to nine weeks. Reagents Chemicals were obtained from the following sources: NG-nitro-L-arginine methyl ester (L-NAME), NG-nitro-Darginine methyl ester (D-NAME), L-arginine, adenosine diphosphate (ADP) and PKH26 from Sigma Chemical (St. Louis, Missouri, USA); RPMI 1640 medium from Gibco (Grand Island, New York, USA); fetal bovine serum from JRH Biosciences (Lenexa, Kansas, USA); zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG) from Porphyrin Products (Logan, Utah, USA). Cells The colon26 murine colonic adenocarcinoma cell line [26] was kindly provided by Dr A. Hagiwara (Departmen of Digestive Surgery, Kyoto Prefecutural University of Medicine, Japan). The cells were maintained in RPMI 1640 medium with 10% fetal bovine serum supplemented with antibiotics. Cell cultures were maintained in plastic flasks in a humidified, CO2 atomosphere at 37 ◦ C. We used only low-passaged cells (three to nine passages) in all of our experiments. Experimental pulmonary or liver metastasis All of the experimental procedures were performed with the approval of the Committee for Animal Research, Kyoto Prefectural University of Medicine. In the pulmonary metastasis experiment, 2 × 104 colon 26 cells in 0.5 ml plasmafree Hanks’ balanced salt solution (HBSS) were inoculated through a tail vein. In the liver metastasis experiment, 1×106 colon26 cells in 0.1 ml HBSS were injected intrasplenically and the animals were splenectomized 5 min later under anesthesia. L-NAME (2.5 mg/9.2 µmol) alone or D-NAME (2.5 mg/9.2 µmol) alone or L-NAME (2.5 mg/9.2 µmol) and L-arginine (1.6 mg/9.2 µmol or 16 mg/92 µmol) was dissolved in a volume of 0.2 ml saline solution and then
Retention of PKH26-labeled colon26 cells in the lung or liver
Time course analysis of serum NO concentration in mice L -NAME or D -NAME was injected into mice according to the schedule described above. Blood was obtained 3, 11 and 27 h after the start of their administration. Following separation, serum was diluted with a 3-fold volume of nitrite/nitrate-free distilled water and filtered through a Centricon-10 concentrator tube (Amicon Inc., Massachusetts, USA) with a molecular exclusion of 10 kDa by centrifugation at 20,000 g. As described by the manufacturer, NO was assessed in serum by measuring its oxidation products, nitrite and nitrate, using a fluorometric assay (DOJINDO, Tokyo, Japan).
Platelet aggregation The mice had taken D-NAME or L-NAME 3 h and 1 h prior to blood collection. Blood was obtained from the inferior vena cava under anesthesia. Blood was immediately mixed with 1/9 volume of 3.2% trisodium citrate dihydrate. The mixture was centrifuged at 150 g for 10 min, and supernatant was obtained as platelet-rich plasma (PRP). The precipitate was centrifuged at 2,000 g for 10 min, and supernatant was obtained as platelet-poor plasma (PPP). Platelet aggregation was measured with a platelet aggregometer using 180 µl of a sample PRP. For induction of aggregation, 20 µl of ADP (final concentration 5 µM) was used. Platelet aggregation was monitored by determining the percentage of light transmission of a sample compared with that of a reference (PPP).
Constitutive nitric oxide synthase, constitutive heme oxygenase and cancer metastasis
Figure 1. Effects of NO-/CO-related compounds on pulmonary metastasis. (a) D-NAME (10 mg/body), L-NAME (10 mg/body), L-arginine (16 mg/body) or L-NAME and L-arginine were injected into mice 3 h and 1 h before and 1 h and 3 h after injection of tumor cells. (n = 10 per group) (b) ZnDPBG (0.083 mg/body) or vehicle was injected into mice 1 h before tumor inoculation. (n = 6 per group). The lungs were removed 18 days after tumor inoculation, and tumor nodules were counted. The data are presented as the mean ± standard error. ∗ P < 0.05 as compared with the group treated with D-NAME. † P < 0.05 as compared with the group treated with L-NAME.
Statistical analysis Differences between the results of experimental treatments were analyzed with a Scheffe-type multiple comparison test and analysis of variance (ANOVA) using the Stat View-J 5.0 computer software. Statistical significance was predetermined as P < 0.05.
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Figure 2. Effects of NO-/CO-related compounds on liver metastasis. (a) D-NAME (10 mg/body), L-NAME (10 mg/body), L-arginine (16 mg/body) or L-NAME and L-arginine were injected into mice 3 h and 1 h before and 1 h and 3 h after injection of tumor cells. (n = 5 per group). (b) ZnDPBG (0.083 mg/body) or vehicle was injected into mice 1 h before tumor inoculation. (n = 10 per group). The liver was removed 11 days after tumor inoculation, and liver weight was measured as an index of metastasis. The data are presented as the mean ± standard error. ∗ P < 0.05 as compared with the group injected with vehicle only.
lungs in the L-NAME-treated group and 107.90 (range, 77– 137) in the D-NAME-treated group. Differences between the values were statistically significant (P < 0.05). The Larginine alone did not produce significant effects on pulmonary metastasis compared with the D-NAME-treated group. The effect of L-NAME was prevented when L-arginine was injected simultaneously, and this was dose-dependent. On the other hand, the treatment with ZnDPBG did not affect the number of pulmonary metastases (Figure 1B).
Results
Liver metastasis model
Pulmonary metastasis model
Large confluent tumors were present all over the liver in all groups 11 days after tumor inoculation. As demonstrated in Figure 2A, the treatment with L-NAME did not affect the weight of the liver compared with the D-NAME-treated group, but the treatment with ZnDPBG increased the mean
Metastatic nodules in the lungs were present in all groups 18 days after tumor inoculation. As demonstrated in Figure 1A, there were 152.00 (range, 88–197) metastatic nodules in the
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T. Ishikawa et al. Table 1. Effects of D-NAME or L-NAME on the retention of PKH26-labeled colon26 cells.
Table 3. Effects of D-NAME or L-NAME on ADP-induced platelet aggregation.
Treatment
Number of PKH26-labeled tumor cells (/mm2 ) Lung Liver
Treatment
Number of mice
Maximal aggregation (%)
D -NAME
D -NAME
1.93 ± 0.27 (n = 6) 5.10 ± 0.25 (n = 6)∗
L-NAME
n=5 n=5
34.80 ± 6.68 44.60 ± 11.25
L-NAME
4.67 ± 1.04 (n = 5) 7.39 ± 1.58 (n = 5)
D -NAME
of L-NAME were injected into mice 3, 1 h before and 1, 3 h after 0 h. At 24 h after 0 h, the number of PKH26labeled tumor cells were counted using a fluorescent microscope. The data are presented as mean ± standard error. ∗ P < 0.05, significant difference from the value in mice treated with vehicle.
D -NAME or L-NAME were injected into mice 3 and 1 h before platelet aggregation was measured. Maximal aggregation is shown by the percentage of light transmission of a sample compared with a reference. The data are presented as mean ± standard error.
Table 2. Effects of ZnDPBG on the retension of PKH26-labeled colon26 cells. Treatment
Number of PKH26-labeled tumor cells (/mm2 ) Lung Liver
Vehicle ZnDPBG
10.07 ± 0.39 (n = 3) 11.20 ± 0.59 (n = 3)
3.67 ± 0.24 (n = 3) 8.00 ± 1.70 (n = 3)∗
Vehicle or ZnDPBG was injected into mice 1 h before tumor inoculation intrasplenically. The number of PKH26-labelled tumor cells were counted using a fluorescent microscope 24 h after tumor inoculation. The data are presented as mean ± standard error. ∗ P < 0.05, significant difference from the value in mice treated with vehicle.
weight of the liver significantly compared with that of the control group (Figure 2B). Retention of PKH26-labeled colon26 cells in the lung or liver The L-NAME-treated group showed a mean of 5.10 tumor cells/mm2 in the lung, whereas the D-NAME-treated group exhibited a mean of 1.93 tumor cells/mm2 (Table 1). Differences between means were statistically significant (P < 0.05). However, in the liver, retention of tumor cells in mice treated with L-NAME was not increased significantly compared with the control group treated with D-NAME (Table 1). On the other hand, the means of tumor cells in the liver were 8.00 tumor cells/mm2 in the ZnDPBGtreated group and 3.67 tumor cells/mm2 in the control group (Table 2). Differences between means were statistically significant (P < 0.05), but ZnDPBG did not increase pulmonary metastasis significantly, compared with the control group (Table 2). Platelet aggregation The mean of maximal aggregation induced by ADP in the L -NAME-treated group was 44.60% and 34.80% in the D NAME-treated group. Differences between the values were not statistically significant (Table 3). The time course analysis of serum NO concentration in mice Treatment with D-NAME did not affect values of serum NO metabolites compared with non-treated mice. Injection of LNAME reduced the serum concentration of NO metabolites
Figure 3. Time course analysis of serum concentration of nitrite plus nitrate. Drugs were injected into mice four times every 2 h. At 3, 8 and 27 h after the first drug injection, the serum concentration of nitrite plus nitrate was measured. D-NAME treatment did not affect the serum concentration of nitrite plus nitrate compared with the values of non-treated mice. The data are presented as the mean ± standard error of measurements from 5–7 separate sera. ∗ P < 0.05 compared with values of D-NAME group. † P < 0.01 compared with values of D-NAME group.
at 3 and 8 h after the start of drug injection compared with that of D-NAME. However, the values returned to normal level at 27 h (Figure 3).
Discussion The present study provides evidence that constitutive NOS and HO influence experimental metastasis and that their effects vary depending on the target organ. We confirmed reduction of the serum concentration of NO metabolites after administration of L-NAME and the concentration returned to normal levels 27 h after the first administration. These findings revealed that reduction of endogenous NO in the early phase induced by administration of L-NAME influenced the outcome of experimental metastasis. Although previous studies revealed many physiological functions of CO and we proposed that the potentiation of the liver metastasis induced by administration of ZnDPBG is mainly attributable to inhibition of CO, we must take into account the fact that the product of HO reaction contains not only CO but also biliverdin and ferric iron. Both NO and CO regulate many cell functions by activating soluble guanylate cyclase, a cyclic guanosine monophosphate (GMP) -producing heme enzyme. Although the reactivity of NO with heme proteins is much higher than that
Constitutive nitric oxide synthase, constitutive heme oxygenase and cancer metastasis of CO [22], inhibition of NO did not affect liver metastasis in this study. Suematsu et al. reported that CO is an endogenous modulator of hepatic sinusoidal perfusion [24, 27]. They indicated that under unstimulated conditions endogenous NO production was unlikely to be involved in vasorelaxation in hepatic sinusoids [28]. They proposed that the concentration of NO in a local compartment (e.g., the Disse space) might be extremely low compared with that of CO because NO and superoxide counteract each other in the liver to maintain a normal balance between them. Under these circumstances a nonradical monoxide such as CO might serve as an effective agonist for cGMP up-regulation. In the present study, our data is consistent with the evidence that CO plays a central role in modulating hepatic microcirculation. Although the mechanisms of the inhibitory effects of constitutive NOS and HO on tumor metastasis have not been clarified, we do know that both NO and CO are capable of influencing tumor cell arrest in capillaries through several mechanisms. Since both NO and CO are potent vasodilatators, their inhibition would markedly reduce the diameter of arterioles and venules. Therefore, an increase in metastasis induced by L-NAME or ZnDPBG might be related to a decrease in the diameter of these vessels, which enhances tumor cell arrest in capillaries. Many studies have implicated platelets in the hematogenous dissemination of tumors [29–31]. NO is an inhibitor of platelet adhesion and aggregation [9, 10, 15]. Marek et al reported that a human colorectal adenocarcinoma cell line with lower activity of NO synthase was a more potent inducer of platelet aggregation, and the generation of NO by tumor cells was inversely correlated with metastatic potential [25]. It has been reported that CO also inhibits platelet aggregation [21]. Thus inhibition of endogenous NO or CO production appears to increase platelet aggregation and to promote metastasis via enhanced tumor cell arrest in capillaries. However, in this study L-NAME showed no significant effects on maximal platelet aggregation induced by ADP. Therefore the promotable effects of L-NAME or ZnDPBG on cancer metastasis may not be produced via enhancing platelet aggregation. Recent evidence suggests that polymorphonuclear neutrophils serve as a carrier to assist tumor cell transendothelial migration [32]. Previous studies reported that NO inhibited neutrophil adhesion to the post capillary venular endothelium [33, 34]. It has been reported that HO induction in the microvascular endothelium significantly attenuates oxidantelicited P-selectin translocation, resulting in the reduction of leukocyte adhesion [35]. These results indicate that NOS and HO could inhibit tumor metastasis by reducing the tumor cell interaction with leukocytes. It has been recently proposed that NO attenuates adhesion molecule expression on human endothelial cells. Takahashi et al. reported that the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on human umbilical vein endothelial cells induced by IL-1β was significantly inhibited in the presence of an NO donor [17]. These adhesion molecules played an important role in tumor cell adhesion to en-
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dothelial cells [36]. Kong et al. indicated that an exogenous NO donor significantly reduced tumor cell adhesion to naïve as well as lipopolysaccharide (LPS)-treated isolated postcapillary venules. L-arginine, a NO precursor, attenuated the tumor cell adhesion to LPS-activated postcapillary venules, and this effect of L-arginine was reversed by administration of L-NAME [16]. On the other hand, Khatib et al. reported that intrasplenic/portal inoculation of H59 cells from Lewis lung carcinoma sublines triggered a rapid increase in cytokines such as IL-1 and TNF-α, resulting in the induction of E-selectin expression in the liver [37]. We propose that the increase in metastasis by L-NAME administration may be related to the effects of endogenous NO on adhesion molecule expression in endothelial cells in target organs. In summary, our results suggest that constitutive NOS attenuates experimental pulmonary metastasis by inhibiting the retention of tumor cells in microcirculatory systems but does not attenuate liver metastasis. On the other hand, constitutive HO attenuates liver metastasis by inhibiting the retention of tumor cells but does not attenuate pulmonary metastasis. In this study, we proposed that L-NAME and ZnDPBG might mainly affect host cells, such as endothelial cells and platelets. However, these drugs could affect not only host cells but also cancer cells because we confirmed inducible NOS (iNOS) and HO-1, 2 expression in colon26 cells by RT-PCR (data not shown). Regarding the influence of constitutive NOS and HO on cancer metastasis, not only a change of hemodynamics but also the interaction between cancer cells and other cells, such as endothelial cells, platelets and leukocytes, should be considered. Further investigation is obviously required to clarify the mechanism of the inhibitory effects of constitutive NOS and HO on tumor metastasis.
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