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A model to study the effectiveness of potential chemopreventive agents that inhibit neoplastic process by different mechanisms has been used to test the efficacy of seven nitric oxide synthase (NOS) inhibitors. Five selective inducible NOS (iNOS) inhibitors: S-methyl isothiourea (S-MITU), S-2-aminoethyl isothiourea (S-2-AEITU), S-ethyl isothiourea (S-EITU), aminoguanidine (AG), 2-amino-4-methyl pyridine (2-AMP), and two non selective general NOS inhibitors: l-N6-(1-iminoethyl) lysine (IEL) and Nω-nitro-l-arginine (NNLA), were tested for efficacy against a carcinogen, benzo[a]pyrene (B[a]P)-induced primary rat tracheal epithelial (RTE) cell transformation assay. RTE cells were treated with B[a]P alone or with five nontoxic concentrations of an NOS inhibitor and the resulting foci at the end of 30 days were scored for inhibition of transformation. The results indicate that all three isothiourea compounds inhibited B[a]P-induced RTE foci in a dose-dependent manner. S-AEITU was the most effective inhibitor with an IC50 (the molar concentration that inhibits transformation by 50%) of 9.1 µM and 100% inhibition at the highest dose tested (30 µM). However, both S-EITU and S-MITU showed a maximum percent inhibition of 81% and 100% at 1 mM with an IC50 of 84 and 110 µM, respectively. 2-AMP did not show any dose-dependent response, but was highly effective (57% inhibition) at an intermediate dose of 30 µM and an IC50 of 25 µM. Similar to thiourea compounds, AG exhibited good dose-dependent inhibition with a maximum inhibition of 86% at 1 mM. NNLA and IEL were negative in this assay. Based on the IC50 values, NOS inhibitors were rated for efficacy from high to low as follows: S-2-AEITU<2-AMP<AG<S-MITU<S-EITU. The data from this study identify NOS inhibitors as a novel class of chemopreventive agents that can be developed for lung cancer prevention.
A significant number of compounds that theoretically have chemopreventive potential can be isolated from natural substances or synthesized. A logical approach must be developed to efficiently evaluate the chemopreventive properties of each compound. Although the effectiveness of any chemopreventive agent depends on its properties in the living animal, in vivo evaluation is very expensive and time consuming. In vitro test systems are ideal for investigating target organ specificity. One such in vitro method that uses primary cultures of rat tracheal epithelial (RTE) cells was previously developed to analyze and quantitate the process of transformation in the respiratory system [1–5]. This protocol was adapted for studying the inhibition of chemically induced transformation and has been expanded to test potential chemopreventive compounds belonging to different chemical classes and biologic activity [6–8]. An extensive evaluation of test data from different in vitro assays generated for 9 years to predict the efficacy in animal model reveals that RTE assay had the highest correlation to hamster lung model and had the highest predictive value of 76% among the assays .
RTE cells provide a relevant model for studying the effectiveness of chemopreventive agents that inhibit the neoplastic process by different mechanisms. The RTE cell focus inhibition assay has been shown to be sensitive to several classes of chemopreventive agents. For example, retinoic acid (RA) has been reported to consistently inhibit in vitro transformation of RTE cells by carcinogen exposure . Steele et al.  showed that RA at a nontoxic concentration could inhibit benzo[a]pyrene (B[a]P)-induced transformation of RTE cells. Using the current protocol for the RTE assay , our laboratory has analyzed responses of a variety of agents tested within the RTE assay in relation to their mechanisms of action and chemical classes. For example, analysis of data from 90 compounds revealed that antioxidants/free radical scavengers, including different isomers of RAs and their derivatives, are a major mechanistic class of agents that are highly effective in the RTE transformation assay . In addition, comparative chemopreventive efficacy of different forms of tea extracts and other polyphenols were identified using the same transformation system and several mechanism-based cell culture assays [12,13]. In the current study, the efficacy of a new class of antioxidants, nitric oxide synthase (NOS) inhibitors, were identified as effective agents for the first time using a B[a]P-induced transformation model of primary RTE cells.
Aminoguanidine (AG), 2-amino-4-methyl pyridine (2-AMP), l-N6-(1-iminoethyl) lysine (IEL), S-methyl isothiourea (S-MITU), Nω-nitro-l-arginine (NNLA) were obtained from Sigma Chemical (St. Louis, MO). S-2-aminoethyl isothiourea (S-2-AEITU) and S-ethyl isothiourea (S-EITU) were obtained from TCI America (Portland, OR). Test agents were dissolved in media on dimethyl sulfoxide.
The RTE cultures were grown and maintained in a modified Ham's F-12 medium as reported by Wu et al.  and incubated at 37°C in an atmosphere of 5% carbon dioxide (CO2) in air. Complete growth medium consists of Ham's F-12 medium mixed in equal proportion with 3T3-conditioned Dulbecco's minimal essential medium (DMEM) containing 2% fetal bovine serum (FBS). This mixture was supplemented with 10 µg insulin/ml, 5 µg human transferrin/ml, 0.2% bovine hypothalamus extract, 1 µM hydrocortisone, 2 µg fungizone/ml, 20 µg gentamycin/ml, and a FBS concentration of 1%.
RTE cells were isolated from 8- to 12-week-old, viral antigen- and specific pathogen-free, male Fischer 344 (F-344) rats (Charles River, Raleigh, NC). The animals were sacrificed by CO2 asphyxiation, the tracheae were excised surgically, the tracheal lumens were filled with Joklik's minimal essential medium (Life Technologies, Rockville, MD) and 1.0% protease (Sigma Type XIV) and the tracheae were incubated overnight at 4°C. The lumens were rinsed with DMEM plus 10% FBS to collect the primary RTE cells, the cells were filtered through a 100-µm-pore nylon mesh, collected by centrifugation, counted with a hemocytometer, and resuspended in complete medium. After determining viability by trypan blue exclusion the single-cell suspensions were plated onto dishes previously coated with a collagen film (600 µg/60-mm dish) at 2x104 cells per dish.
To select appropriate nontoxic doses for inhibition of transformation, an initial cytotoxicity test was conducted for all NOS inhibitors. The dose selection assay procedures have been published previously . Briefly, RTE cells plated (2x104 cells per 60-mm dish) in collagen-coated dishes (600 µg/dish) for a 24-hour attachment period, were exposed to a carcinogen, B[a]P (10 µg/ml), plus the highest soluble concentration of NOS inhibitors in medium or solvent up to 1 mM (1 mg/ml) plus four log dilutions of that concentration. After another 24 hours, the B[a]P-containing medium was replaced with complete culture medium containing either chemopreventive agent or appropriate solvent. After 5–7 days, surviving colonies of >50 cells were counted using an automatic colony counter and the relative cytotoxicity was determined by measuring reduction in colony-forming efficiency (CFE) in test agent-treated compared to untreated (solvent) control.
A detailed version of the assay procedures is described elsewhere . Briefly, the RTE cells were plated and treated with B[a]P as described before. From the initial cytotoxicity test, a concentration of the NOS inhibitors that reduces CFE to approximately 80% of control levels (i.e., a 20% or lower reduction compared to controls) was used as the highest concentration plus four half-log dilutions for the transformation inhibition assay. Culture medium containing fresh test agent was changed twice weekly. Between days 5 and 7, a set of replicate cultures per group were scored for CFE and analyzed for potential toxicity of test doses. After day 15, the cultures were exposed to reduced serum conditions to increase the selection pressure against normal cells and to reduce the background rates of transformation in the control cultures. Because transformed RTE cells require less serum and conditioned medium to grow, after 14 days the conditioned medium was reduced to one third , making the final concentration of serum to be 0.5%. This reduction allows normal cells to senesce and provides easier identification and measurement of the transformed foci. At day 30, the cultures on dishes were fixed in methanol, stained with Giemsa, and scored for transformed colonies. Three types of transformed colonies (class I, II, and III) were identified on the dishes according to the size of the colony, the size of the cells, the number of cells per colony, the staining density of the colony, as well as the morphologic characteristics of the cells . Previous studies  have shown that class III transformed colonies or foci that have greater than 2500 cells/mm2 progress to form tumors when injected into nude mice. A very high percentage of class II foci (1300–2500 cells/mm2 also became tumorigenic. Class I foci (<1300 cells/mm2) usually do not progress to tumorigenicity. Therefore, only class II and III foci are considered as evidence of morphologic transformation in this assay. The transformation frequency was calculated by dividing the number of class II+class III foci per dish by the surviving colony-forming units per dish. This procedure corrects for any cytotoxicity of the chemopreventive agent. The percent reduction in transformation frequency induced by the test agent was to be calculated for each group by the formula: ΔTF=(TFCA-TFCP)/TFCAx100, where TFCA is the transformation frequency of the carcinogen alone (minus solvent control frequency) and TFCP is the transformation frequency of the carcinogen plus chemopreventive (NOS)-treated cells (corrected for solvent control). The comparison of data from control and experimental groups was analyzed, and an agent was considered positive if two or more concentrations inhibit carcinogen-induced foci formation by 20% or more.
Abnormal synthesis of NO by both constitutive (neuronal and endothelial) and inducible (type II) isoforms of NOS (iNOS) has been implicated in a variety of human diseases including circulatory shock. NOS inhibitors that cause nonspecific inhibition of NO formation can result in side effects by inhibiting the constitutive forms of NOS that are essential for the various physiological functions of NO. To circumvent this problem, selective NOS inhibitors such as S-MITU, S-2-AIETU, and S-EITU, which belong to thiourea class of compounds, were developed. They are well known for their selective NOS inhibition especially the type II (inducible) NOS [14–16]. Studies also indicate that in addition to inhibition of NOS, type II NOS inhibitors have several other potential therapeutic applications, including treatment of sepsis, diabetes, and autoimmune diseases . A number of in vivo studies have indicated the effectiveness of NOS inhibitors by targeting NOS including iNOS as good targets for chemoprevention of colon cancer in animals [17,18]. An in vitro system of primary mouse macrophage culture stimulated with IFN-γ or lipopolysaccharide has also been used to identify novel triterpenoids as selective iNOS inhibitors . In the RTE assay, all three iNOS compounds were highly efficacious with dose-dependent inhibition ranging from 50% to 100% (Figure 1). Based on the IC50 values, S-2-AEITU was more efficacious (9.1 µM) than S-MITU (110 µM) or S-EITU (84 µM) (Table 1). Although both S-MITU and S-EITU have high IC50 values, they were also nontoxic up to 1 mM. Studies show that S-MITU significantly inhibited the nitric oxide formation in immunostimulated culture of macrophages and vascular smooth muscle cells with IC50 at 6 and 2 µM, respectively, and was 10-to 30-fold more potent when compared with other inhibitors. It was used in the therapy of circulatory shock . S-EITU, another iNOS inhibitor, decreased the lipopolysaccharide-induced plasma nitrite and nitrate concentration by 95% in rats with an ED50 concentration of 0.4 mg/kg. It was also found to suppress the internucleosomal DNA cleavage in pancreatic beta cells induced by NOS . Data from another in vivo study indicated that S-EITU (0.1 mg/kg) significantly increased the survival time and rate in phenylephrine-induced splanchnic artery occlusion shock in rats .
2-AMP, belonging to another class of type II NOS inhibitor, was as effective as the thiourea compounds with a maximum inhibition of 57% at 30 µM and an intermediate IC50 value of 25 µM (Figure 2). Investigations suggest that 2-AMP can inhibit the catalytic activity NOSII enzymes both in vitro and in vivo. 2-AMP can also inhibit lipopolysaccharide-induced elevation of plasma nitrate in rats after either subcutaneous (ID50=0.3 mg/kg) or oral administration (ID50=20.8 mg/kg) . Early studies showed that 2-AMP facilitated neuromuscular transmission by the inhibition of cholinesterase .
Aminoguanidine (AG), an irreversible iNOS inhibitor, was also effective in the RTE assay, with an intermediate IC50 of 26 µM (Figure 2) similar to 2 AMP. AG and its derivatives have been shown to have anticancer and antiviral activities. Studies indicate that some of the derivatives are effective in inhibiting Rous sarcoma virus transformation of chick fibroblast in vitro, P388 leukemia in mice  and proliferation of Ehrlich ascites cells grown in culture . Other studies show that eight derivatives of AG were effective against human leukemic cells in vitro, mainly by inhibiting ribonucleotide reductase activity with IC50 ranging from 2.95 to 121 µM . Recent studies indicate that AG also can reverse inhibition of glucose-induced angiogenesis as a part of wound healing process in simulated diabetic rat model .
Other NOS inhibitors, IEL and NNLA, which are also amino acid derivatives, were not efficacious in the RTE assay even at 1 mM concentrations (Figure 3). NNLA is a nonselective NOS inhibitor. Studies indicate that it significantly inhibited the nitrite accumulation in murine breast cancer cell line . However, as it is a nonspecific inhibitor of both constitutive NOS and iNOS, it may not be a desirable compound to be developed for lung cancer prevention. Further, in vivo studies with a pulmonary metastasis model have indicated that l-NAME, a methyl ester of NNLA, increased the number of metastases induced by Lewis lung carcinoma and B16 melanoma cells . IEL, a nontoxic ineffective compound in the RTE transformation system (<20% inhibition), potentially inhibited the activity of iNOS in primary macrophage of intact cells and during the infection of mice with a NO sensitive parasite, Leishmania major .
NO is an important endogenous mediator in regulating normal airway function. However, NO synthesis mainly by the iNOS isoform is also implicated in the pathophysiology of inflammatory airway diseases mediated by various cytokines . The role of NO in tumorigenesis is complex with increased NOS expression implicated in tumor progression as well as metastasis [32–38]. When NO is produced at high concentrations mainly by iNOS alone, it has both cytotoxic and cytostatic properties leading to DNA damage, an initial step in carcinogenesis. However, at conditions of low concentrations generated mostly by constitutive NOS isoforms, tumor growth is enhanced by NO's antiapoptotic effects along with its ability to induce angiogenesis . Therefore, it is critical to select an NOS inhibitor that is specific for the inducible isoform in cancer prevention or treatment scenario. The data from this study identify a new class of compounds, NOS inhibitors, especially the iNOS inhibitors (in addition to their other desirable biologic activities described above) as effective chemopreventive agents that can be developed as agents for lung cancer prevention. Further preclinical studies of these inhibitors in in vivo lung cancer models would be of great interest.
1This work was funded (N01-CN-85044) by the Division of Cancer Prevention, National Cancer Institute, USA.