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Mol Cancer Ther. 2003;2:1285-1293
© 2003 American Association for Cancer Research

Nitric oxide does not mediate but inhibits transformation and tumor phenotype

Arindam Dhar1, June M. Brindley1, Cristi Stark1, Michael L. Citro3, Larry K. Keefer2 and Nancy H. Colburn1

1 Gene Regulation Section, Basic Research Laboratory; 2 Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick, Frederick, MD; and 3 Basic Research Program, SAIC-Frederick, Frederick, MD

Requests for Reprints: Arindam Dhar, Building 567, Room 180, National Cancer Institute at Frederick, Frederick, MD 21702. Phone: (301) 846-6756; Fax: (301) 846-6907. E-mail: adhar{at}ncifcrf.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although inducible nitric oxide synthase (iNOS) and nitric oxide (NO) are implicated in tumor pathology, their role in the early stages of carcinogenesis is not well defined. Tumor necrosis factor {alpha} (TNF{alpha}) induces iNOS and NO production in transformation-sensitive JB6 P+, but not in transformation-resistant JB6 P-, mouse epidermal cells. We tested the hypothesis that iNOS, by generating NO and reactive nitrogen species, mediates tumor promoter-induced transformation. Specific [N-[3-(aminomethyl)benzyl]acetamidine (1400W)] and non-specific (N{omega}-methyl-L-arginine) iNOS inhibitors significantly reduced TNF{alpha}-induced NO production in P+ cells but both iNOS inhibitors enhanced TNF{alpha}-induced anchorage-independent transformation, thus ruling out a mediator role and suggesting an inhibitor role for NO. Independent support for an inhibitor role came from the observation that the NO donor [(Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA/NO)] inhibited TNF{alpha}- and 12-O-tetradecanoylphorbol-13-acetate-induced transformation. DETA/NO treatment also suppressed tumor phenotype in tumorigenic JB6 RT101 (Tx) cells. Higher concentrations of DETA/NO induced apoptosis. The transformation inhibitory effect of lower DETA/NO concentrations may be attributable in part to inhibition by NO of NF-{kappa}B-dependent but not of AP-1-dependent transcription. In conclusion: (a) induction of iNOS and NO production does not mediate but actually prevents tumor promotion; (b) iNOS inhibitors enhance the transformation response, and therefore appear not to be appropriate as chemoprevention agents; and (c) NO has both chemopreventive and tumoricidal effects, suggesting promise in cancer chemoprevention and therapy.

Key Words: iNOS • nitric oxide • NF-{kappa}B • AP-1 • tumor promoter


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The generation of reactive oxygen and nitrogen species (ROS/RNS) has been implicated in the pathogenesis of cancer and degenerative diseases. Recent evidence suggests that oxidative and nitrosative stress, resulting from environmental exposure or from stimulus-induced intracellular enzymatic activity, alters transcriptional response by posttranslational modification of transcription factors (1–4). One of the key elements in this process is the generation of nitric oxide (NO) by the intracellular enzymatic activity of the nitric oxide synthase (NOS) (3, 5, 6). Of the three isoforms of the enzyme—endothelial (eNOS), neuronal (nNOS), and inducible nitric oxide synthase (iNOS)—the iNOS is induced by a large number of external stimuli and may lead to high levels of NO (5, 6). While low concentrations of NO alter transcription factor activity (mainly NF-{kappa}B and AP-1) and act as an antioxidant, high concentrations of NO can damage DNA and proteins (2, 7, 8). NO-induced alteration in cellular function can lead to cell death, degenerative disorders, or stimulation of tumor growth and metastasis (9, 10).

The role of iNOS in tumor promotion and tumor progression is not clear. Elevated expression of iNOS is associated with premalignant and malignant lesions from breast (11), stomach (12), colon (13), lungs (14), prostate (15), melanocytes (16), and skin (17). In the case of breast and gynecological primary tumor tissue, iNOS enzymatic activity showed positive correlation with tumor phenotype (11, 18). In the colon, iNOS contributes to progression of adenoma to carcinoma, and inhibition of iNOS prevents intestinal polyposis (19–21). Other studies show that either induction or overexpression of iNOS suppresses tumorigenicity or metastasis in melanoma and renal carcinoma cell lines (22–24). While iNOS may play a role in the maintenance of tumor phenotype, little is known about its functional significance during cancer induction, particularly during tumor promotion, a rate-limiting stage in multistage carcinogenesis that yields premalignant lesions.

NO is reported to have mutagenic properties, especially on long-term exposure which occurs in cells during chronic infection (25, 26). NO mutates p53 during colon carcinogenesis (27) and alters p53 function by nitration of tyrosine (28, 29). However, a causal relationship of NO to carcinogenesis has not been established. In an in vitro model of chemical carcinogenesis, NO production has been associated with transformation of C3H10T1/2 fibroblasts (30). In an in vivo model of two-stage skin carcinogenesis, an NO donor (nitroglycerine) inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion in mouse skin (31). These apparently conflicting findings suggest that the role of NO in tumor promotion needs to be further defined.

The JB6 murine epidermal cell model has been predictive in identifying molecular targets and defining their role in tumor promotion (32). The JB6 model consists of clonal variants that are transformation-sensitive (P+) or -resistant (P-). On treatment with tumor promoters like TPA, tumor necrosis factor {alpha} (TNF{alpha}), or epidermal growth factor (EGF), the P+ cells respond with induction of anchorage-independent growth in soft agar and tumorigenicity on mouse xenograft (33, 34). The P- cells do not show transformed phenotype on treatment with tumor promoters. Among the major mediators of the transformation-sensitive phenotype of P+ cells are the ERK/AP-1 and NF-{kappa}B pathways (35–41). ROS play a significant role in inducing transformation of P+ cells. Antioxidant enzymes such as superoxide dismutase block the ROS-dependent JB6 transformation response (42, 43), paralleling mouse skin tumor promotion studies (44). A recent study has shown that TNF{alpha} induces iNOS in promotion-sensitive P+ cells but not in promotion-resistant P- cells in a coculture assay (45), suggesting that iNOS might be functionally significant in the transformation of P+ cells and that a lack of iNOS induction might contribute to the resistance of P- cells.

In the present study, we used iNOS inhibitors and a NO donor to test the hypothesis that iNOS activation and NO generation are required for TNF{alpha}-induced transformation. Specific [N-[3-(aminomethyl)benzyl]acetamidine (1400W)] and non-specific (N{omega}-methyl-L-arginine) inhibitors of iNOS suppressed TNF{alpha}-induced NO production in P+ cells. However, the iNOS inhibitors not only failed to block, but actually enhanced TNF{alpha}-induced transformation of P+ cells. Conversely, treatment with the NO donor (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA/NO) inhibited the transformation response and suppressed tumor phenotype in the JB6 model. These findings show that iNOS induction and subsequent NO production are not only not causal but rather protect against tumor promotion and suppress tumor phenotype by a mechanism that may involve inhibiting NF-{kappa}B-dependent transcription or inducing apoptosis or both.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
Mouse epidermal JB6 cells have been described previously and were grown according to protocol (35). Briefly, JB6 P+ cells were cultured at 36°C in EMEM (Bio Whittaker, Frederick, MD) supplemented with 4% FBS, 2 mM L-glutamine, and 25 mg/ml gentamycin (Invitrogen, Carlsbad, CA). The reporter cell line consisted of P+ cells stably transfected with an AP-1 promoter-luciferase reporter construct (A9 cells) or NF-{kappa}B promoter-luciferase reporter construct (N3 cells). The promoter-reporter construct has been described in detail previously (40). The A9 and N3 cells were maintained as P+ cells, with G418 selection every 4–6 weeks. The iNOS inhibitors (1400W and N{omega}-methyl-L-arginine) and TPA were obtained from Alexis Biochemicals (Lausanne, Switzerland) and TNF{alpha} from PeproTech Inc. (Rocky Hill, NJ). DETA/NO was a kind gift from Dr. Joseph A. Hrabie, Laboratory of Chemical Carcinogenesis, NCI at Frederick, Frederick, MD. The iNOS inhibitors and NO donor were dissolved in water.

Reverse Transcription-PCR for iNOS
JB6 P+ cells, plated at 5 x 104 cells/well in six-well tissue culture dishes, were serum-starved for 24 h [EMEM with 0.2% fetal bovine serum (FBS)] before treatment with TNF{alpha} (10 ng/ml) or TPA (10 ng/ml) for either 6 or 24 h. RNA was isolated as described (46) and reverse transcription (RT) followed by PCR (30 cycles) was carried out with 1 µg of total RNA using the GeneAmp kit and protocol (Applied Biosystems/Roche, Branchburg, NJ). The iNOS primers (F-5'-CCCTTCCGAAGTTTCTGGCAGCAGC-3' and R-5'-GGCTGTCAGAGCCTCGTGGCTTTGG-3') were cDNA specific. RT-PCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.

Cell Growth Assay and Viable Cell Mass
The JB6 P+ cells were plated at 2 x 103 cells/well in 96-well tissue culture plates. After overnight incubation at 36°C, the medium in the well was replaced with fresh medium supplemented with iNOS inhibitors 1400W (10 and 100 µM) or N{omega}-methyl-L-arginine (100 and 500 µM) either alone or in combination with TNF{alpha} (10 ng/ml). After further incubation for 24–72 h, cell number was determined by a modified MTT assay (XTT assay) as per manufacturer's protocol (Roche Applied Sciences, Indianapolis, IN). Cell growth is expressed as increase in colorimetric absorbance (A492 nm–A690 nm reference) measured by a plate reader (Lumimark, Bio-Rad, Hercules, CA). Viable cell mass after DETA/NO treatment was assayed using DETA/NO at 10, 100, and 500 µM concentrations.

TNF{alpha} Treatment for iNOS Induction and NO Production
JB6 P+ cells were plated at 50,000 cells/well in 24-well plates. After washing twice with PBS, the cells were treated with TNF{alpha} (10 ng/ml), iNOS inhibitors 1400W (1, 10, 100 µM) or N{omega}-methyl-L-arginine (100, 500, 1000 µM), or a combination of TNF{alpha} and iNOS inhibitors diluted in EMEM free of FBS and phenol red. During combination treatment, the iNOS inhibitors were added 1 h before TNF{alpha} treatment. After 24 h, the culture supernatants were used for nitrite determination.

Determination of Nitrite Concentration
Because NO is extensively oxidized to nitrite ion in aerobic aqueous media, we used the nitrite concentration as an indicator of the amount of NO produced in cell culture experiments. The nitrite concentration in culture supernatants was determined by the Griess assay using a colorimetric assay kit (Cayman Chemical, Ann Arbor, MI). The medium supernatants were assayed without dilution.

Determination of NO Release from DETA/NO in Culture Medium
The rate of NO release from DETA/NO in buffer and in culture medium was measured in a Thermal Energy Analyzer (TEA) model 502A (from Thermedics, Analytical Instruments Division, Waltham, MA) as previously described (47).

Anchorage-Independent Transformation Assay (Soft Agar Assay)
Soft agar assays for transformation were carried out as described previously (48). Both layers of agar were supplemented with TNF{alpha} (10 ng/ml) or TPA (10 ng/ml) or iNOS inhibitors 1400W (10 and 100 µM) or N{omega}-methyl-L-arginine (100–1000 µM) either alone or as a combination of iNOS inhibitors with TNF{alpha} or TPA. The formed colonies were counted by an automated image analysis system (Image Pro-Plus software, Media Cybernetics, Silver Spring, MD). Transformation response is expressed as colonies per 10,000 cells per 60-mm dish (mean ± SD of four dishes per treatment).

Apoptosis Assay
Cells were plated at 1 x 104 cells per well (100 µl) in 96-well plates. After overnight incubation, the cells were treated with varying concentrations (10, 100, 250, 500 µM) of DETA/NO for 24 h. Apoptosis was measured by an ELISA kit that quantitates fragmented nucleosomes in the cell lysate, as per manufacturer's protocol (Cell Death Detection ELISAPLUS from Roche Applied Sciences, Mannheim, Germany). All experiments were done in triplicate and repeated at least twice. Results are expressed as mean ± SD of triplicates.

Luciferase Reporter Assays
For the AP-1 reporter cell line A9, cells were plated at 2 x 104 cells/well in 24-well tissue culture plates in EMEM with 4% FBS. After overnight incubation, the cells were serum-starved for >24 h in EMEM with 0.25% FBS. The quiescent cells were then treated with TPA (10 ng/ml) or iNOS inhibitors (1400W at 1, 10, and 100 µM or N{omega}-methyl-L-arginine at 100, 500, and 1000 µM) either alone or as a combination of iNOS inhibitors with TPA. Similarly, DETA/NO (at 10 and 100 µM) was substituted for iNOS inhibitors to test for the effect of NO on AP-1-dependent transcription. The cells were lysed 18 h after stimulation, and the lysate was assayed for luciferase activity using Luciferase Assay kit (Promega Corp., Madison, WI) and DYNEX luminometer (DYNEX Technologies, Chantilly, VA). All luciferase activity was expressed as a ratio normalized relative to untreated control (=1). For the NF-{kappa}B promoter-reporter assay, N3 cells were plated and treated the same way as A9 cells, except that TNF{alpha} (10 ng/ml) was used to stimulate NF-{kappa}B activity and cells were lysed after 6 h treatment.

Statistical Analysis
Statistical significance of the differences between groups in each assay was determined by two-tailed Student's t test with unequal variance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In P+ Cells, iNOS Is Induced by TNF{alpha} and not by TPA
Because we were investigating the role of iNOS in transformation response, and P+ transformation is promoted by activation of either AP-1 (by TPA) or NF-{kappa}B (by TNF{alpha}), we examined iNOS induction by these inducers. The murine iNOS promoter lacks a complete AP-1 response element (49). However, TPA induction of the AP-1 response in P+ cells is accompanied by NF-{kappa}B activation (48), and therefore might induce iNOS expression. RT-PCR with iNOS cDNA-specific primers showed that iNOS was induced by TNF{alpha}, but not detectable following TPA treatment of P+ cells (Fig. 1). TNF{alpha} induction of iNOS is an early response (at 6 h) characteristic of NF-{kappa}B activation, and is significantly reduced by 24 h. TPA has no detectable role in induction of iNOS either alone or in conjunction with TNF{alpha}.



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  		Figure 1. iNOS expression is induced by TNF{alpha} in JB6 P+ cells. JB6 P+ cells were treated with TNF{alpha} (10 ng/ml) or TPA (10 ng/ml) or both for 6 or 24 h. RNA was isolated and RT-PCR was carried out with iNOS and GAPDH (control) cDNA-specific primers. RNA from murine macrophage (RAW264.7) cells treated with IFN-{gamma} was used as a positive control (+ve) for iNOS message.

 
Inhibition of TNF{alpha}-Induced iNOS Blocks NO Production in P+ Cells
One prediction arising from the hypothesis that iNOS induction and elevated NO play a causal role in tumor promoter-induced transformation is that iNOS inhibitors should inhibit transformation concomitant with inhibition of NO production. We first examined the effect of iNOS inhibitors on NO generation by TNF{alpha}-induced iNOS in P+ cells. A selective (1400W) and a non-selective (N{omega}-methyl-L-arginine) small molecule inhibitor were used to suppress iNOS activity at doses 4–400 times their in vitro IC50 values (6). While N{omega}-methyl-L-arginine can inhibit iNOS reversibly (Ki = 3.9 µM) and irreversibly (Ki = 2.6 µM) in a dose-dependent manner (50), 1400W is an irreversible or slowly reversible inhibitor (Ki = 7.0 nM) of iNOS (51). Treatment of P+ cells with TNF{alpha} for 24 h resulted in >2-fold induction of NO generation (P <= 0.01) (Fig. 2A). The selective iNOS inhibitor 1400W was effective in blocking TNF{alpha}-induced NO production by 37–78% at concentrations of 1, 10, and 100 µM (P <= 0.05, 0.001, 0.001, respectively). The non-selective iNOS inhibitor N{omega}-methyl-L-arginine blocked TNF{alpha}-induced NO production by 77–83% at concentrations of 100, 500, and 1000 µM (P <= 0.001). Although the inhibitors lowered both basal and TNF{alpha}-induced NO generation, the most significant down-regulation was seen with the induced NO levels.



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  		Figure 2. iNOS inhibitors block TNF{alpha}-induced NO production but significantly enhance TNF{alpha}-induced transformation response. A, specific (1400W) and non-specific (NMA—N{omega}-methyl-L-arginine) iNOS inhibitors block TNF{alpha}-induced NO production in P+ cells. JB6 P+ cells were plated at 50,000 cells/well (24-well plate) in 1 ml of EMEM (4% FBS). After overnight incubation, cells were washed with PBS and treated with TNF{alpha} or iNOS inhibitors or a combination of both as indicated above, diluted in EMEM free of serum and phenol red. When used in combination, iNOS inhibitors were added 1 h before TNF{alpha}. After 24 h treatment, nitrite concentration (as a measure of NO released) was assayed in the supernatant. Each experiment was done in triplicate and repeated twice. (Columns, mean values; bars, SD; P values: *<=0.05, **<=0.01, ***<=0.001 compared to TNF{alpha} treatment, except for a—compared to no treatment control). B, iNOS inhibitors enhance TNF{alpha}-induced transformation of P+ cells. Anchorage-independent cell growth in soft agar (colony formation) as a measure of transformation response was assayed. Columns, mean value of an experiment in triplicate; bars, SD. (P values: *<0.05, **<0.01; compared to treatment with TNF{alpha} alone.) C, NO inhibition does not affect growth of P+ cells in monolayer culture. JB6 P+ cells were plated in 96-well tissue culture plates and treated with iNOS inhibitors 1400W or N{omega}-methyl-L-arginine (NMA), either alone or simultaneously with TNF{alpha} for 72 h. Viable cell number at the end of treatment was determined by a modified MTT assay and expressed as absorbance at 492 nm. Results are representative of experiments done in triplicate.

 
Inhibition of iNOS Enhances Anchorage-Independent Transformation of P+ Cells
To examine the effect of iNOS inhibition on tumor promotion, we used the well-established anchorage-independent transformation assay for P+ cells in the presence and absence of iNOS inhibitors (49). Both inhibitors produced a dose-dependent enhancement of TNF{alpha}-induced transformation response (Fig. 2B). While TNF{alpha} induced colony formation to 3-fold over control (P <= 0.05), 1400W enhanced the response at concentrations of 1, 10, and 100 µM to 1.7-, 2.5-, and 3-fold over TNF{alpha} levels, (P <= 0.05, 0.01, 0.01, respectively). The non-selective inhibitor N{omega}-methyl-L-arginine also enhanced TNF{alpha}-induced transformation at 100, 500, and 1000 µM concentration to 1.9-, 2.2-, 2.8-fold over TNF{alpha} (P <= 0.05, 0.05, 0.01, respectively). There was no significant effect of the inhibitors on TPA-induced transformation of P+ cells (data not shown), indicating specificity of the iNOS inhibitors for enhancing TNF-induced transformation.

Treatment with iNOS Inhibitors Does Not Affect Growth of P+ Cells in Monolayer Culture
We asked whether the enhancement of transformation reflected a general growth enhancement as observed in monolayer culture. Fig. 2C shows that in monolayer culture, TNF{alpha} did not alter growth rate at 72 h as compared to the control. The iNOS inhibitors, either alone or in combination with TNF{alpha}, produced no change in growth rate of P+ cells at concentrations that inhibited TNF{alpha}-induced NO production (Fig. 2C). Neither the generation of NO nor its inhibition by iNOS inhibitors affected the growth of P+ cells in monolayer culture. Thus, iNOS inhibition enhances transformation without affecting growth.

An NO Donor Generates NO in Culture Medium
The observation that iNOS inhibition enhances transformation (Fig. 2B) suggests that NO is an inhibitor of transformation. If this is true, then a chemical NO donor should inhibit transformation. To test this possibility, we used a well-characterized NO donor (DETA/NO) in the anchorage-independent transformation assay. Before use in the cell-based assays, we estimated NO production by DETA/NO in liquid and soft agar culture medium (Fig. 3) over a period of 4 days, a critical time point for determining transformation response (52). In culture medium without DETA/NO, the rate of NO production was below the detection range (1 pmol–5 nmol) of the chemiluminescence method. Six hours after adding DETA/NO (100 µM final concentration), NO release was detected in both media, although the initial values were less for the soft agar. NO showed exponential decay over the first 4 days, reaching a near-zero level between the third and fourth day. The half-life of the prodrug in both media was 9–10 h. We chose a dose range of 10–500 µM DETA/NO, corresponding to the nanomolar concentrations of NO in culture medium. This dose range is reported to be cytoprotective, as opposed to higher doses that produce deleterious effects (53).



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  		Figure 3. DETA/NO generates NO at nanomoles per minute per milliliter for more than 24 h in culture medium. Starting 6 h after adding DETA/NO (100 µM) to liquid or soft agar medium, the rate of NO release at 37°C was measured by chemiluminescence method. In medium without DETA/NO, NO levels were below the detection range (1 pmol–5 nmol).

 
NO Inhibits Transformation of P+ Cells
Anchorage-independent colony formation by P+ cells was assayed after induction with TNF{alpha} (10 ng/ml) or TPA (10 ng/ml) in the absence or presence of increasing concentrations of DETA/NO (10–500 µM). DETA/NO inhibited TNF{alpha}- and TPA-induced transformation in a dose-dependent manner (Fig. 4, A and B). TNF{alpha}-induced transformation showed a greater sensitivity to inhibition by NO, with a 6-fold inhibition at 10 µM (>5-fold, P <= 0.01) and complete inhibition at 100 µM DETA/NO concentration. TPA-induced transformation was not significantly inhibited until 500 µM DETA/NO was used (>3-fold, P <= 0.001).



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  		Figure 4. NO suppresses TNF{alpha}- and TPA-induced transformation in P+ cells. Anchorage-independent colony formation in soft agar was measured with TNF{alpha} (10 ng/ml) (A) or TPA (10 ng/ml) (B) treatment in the absence or presence of increasing concentrations of DETA/NO. Values are average of triplicates; P values: *<=0.05, ***<=0.001; compared to TNF{alpha} treatment in A and TPA treatment in B.

 
NO Suppresses Tumor Phenotype
The transformed (Tx) line (RT101) derived from the JB6 P+ genetic variants demonstrates a tumor phenotype. These cells form anchorage-independent colonies and tumors on mouse xenograft without induction by TNF{alpha} or TPA. To test the effect of NO on tumor phenotype, we assayed anchorage-independent colony formation by Tx cells in the absence or presence of DETA/NO. The Tx cells showed dose-dependent inhibition of colony formation when treated with DETA/NO, with significant inhibition at 100 µM (1.8-fold, P <= 0.01) and 500 µM (2.7-fold, P <= 0.01) (Fig. 5).



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  		Figure 5. NO suppresses tumor phenotype. Tx cells, which express tumor phenotype by forming anchorage-independent colonies without any tumor-promoting stimulus, were assayed for colony formation in soft agar in the absence and presence of DETA/NO. Tx cells' anchorage-independent colony formation was significantly inhibited by DETA/NO in a dose-dependent manner (>10 µM). Values are averages of triplicates; P values: *<=0.05, **<=0.01, ***<=0.001, relative to untreated control.

 
NO Induces Apoptosis
To ascertain whether NO produced a toxic or deleterious effect on the cells at the doses that inhibited transformation or suppressed tumor phenotype, we assayed for apoptosis induction in P+ and Tx cells in the presence of varying concentrations of DETA/NO. Estimation of viable cell number using a modified MTT assay (XTT assay) showed cell loss with 100–500 µM DETA/NO at 24 h (Fig. 6A). By 72 h, a further decrease in viable cells was observed at lower (<100 µM) DETA/NO concentrations. We measured the apoptosis response in P+ and Tx cells after 24 h DETA/NO treatment (Fig. 6B). At doses that decreased viable cell number at 24 h (100–500 µM), DETA/NO induced apoptosis in both P+ and Tx cells. While apoptosis was induced in P+ cells at a lower concentration than in Tx cells, significantly greater apoptosis was induced in the Tx cell lines at >100 µM DETA/NO. An apparent decrease in apoptosis as seen at the 500 µM concentration may be explained as follows. Because apoptosis was assayed by measuring fragmented nucleosomes in cell lysate, early induction of apoptosis at high doses is accompanied by cell rupture and loss of nucleosomes in the supernatant. Subsequent assay for nucleosomes in the supernatant showed a high level of fragmented nucleosomes at 500 µM range (minimal for the other doses—data not shown), indicating that higher doses induce apoptosis faster. In summary, while higher NO concentrations induce apoptosis, the NO concentrations needed for significant inhibition of transformation (10 µM) or tumor phenotype (100 µM) does not increase apoptosis induction.



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  		Figure 6. NO at higher concentrations reduces viable cell number by inducing apoptosis in P+ and Tx cells. A and B, NO reduces viable cell number. Cells were plated at a density of 2 x 103 cells/well in 24-well plates and treated once with DETA/NO at varying concentrations. Viable cell mass was measured using a modified MTT (XTT) assay 24 and 72 h after adding DETA/NO. C, NO induces apoptosis in a dose-dependent manner that parallels effects on viability. Cells were plated at a density of 2 x 103 cells/well in 24-well plates and treated with DETA/NO at varying concentrations. After 24 h treatment, apoptosis was measured in cell lysate by sandwich ELISA to detect fragmented nucleosomes. The drop in apoptotic index at 500 µM DETA/NO treatment was reciprocated by increased fragmented nucleosomes in culture supernatant (data not shown). All values are the average of triplicates; all experiments were repeated at least twice. P values compared to no treatment: **<0.01.

 
NO Inhibits NF-{kappa}B-Dependent, but not AP-1-Dependent, Transcriptional Activation
Because NF-{kappa}B and AP-1 play pivotal roles in the transformation of P+ cells (reviewed in 32), we asked whether NF-{kappa}B- or AP-1-dependent transcription might be targets of NO when it inhibits transformation. TNF{alpha} induces NF–{kappa}B-dependent transcription (and consequent iNOS up-regulation) and transformation in P+ cells. TPA, on the other hand, induces AP-1-dependent transcription and transformation without inducing iNOS. The effect of NO on NF-{kappa}B- and AP-1-dependent transcription was assayed using P+ cell lines stably transfected with either IL-6-NF-{kappa}B-luciferase (N3 cell line) or 4xAP-1-luciferase (A9 cell line) promoter-reporter constructs, respectively. TNF{alpha}-induced NF-{kappa}B-dependent transcription (5-fold induction compared to no treatment, Fig. 7A) was significantly inhibited by DETA/NO treatment at 10 and 100 µM doses (relative to basal, P <= 0.01), while there was no effect on basal activity. In contrast, DETA/NO treatment had no effect on basal or TPA-induced AP-1 activity (Fig. 7B). Thus, NF-{kappa}B, but not AP-1, appears to be a target of NO at the lower concentrations effective in inhibiting transformation.



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  		Figure 7. NO inhibits NF-{kappa}B-dependent, but not AP-1-dependent, transcriptional activation. P+ cells harboring stably integrated NF-{kappa}B-luciferase (A, ±TNF{alpha} 10 ng/ml for 6 h) or 4xAP-1-luciferase (B, ±TPA 10 ng/ml for 18 h) promoter-reporter constructs were simultaneously treated with DETA/NO as shown. Measured luciferase activity in cell lysate (normalized to no treatment) showed significant decrease in NF-{kappa}B-dependent transcriptional activity with DETA/NO even at 10 µM level, while AP-1-dependent transcriptional activity was unaffected. Values are averages of triplicates; P values: **<=0.001; compared to TNF{alpha} treatment in A and TPA treatment in B.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study provides the first clear evidence that NO from both endogenous and exogenous sources can inhibit the rate-limiting tumor promotion stage in a model of epithelial carcinogenesis. Inhibition of endogenous NO production by TNF{alpha}-induced iNOS enhanced the transformation response in JB6 P+ cells. Conversely, production of NO from a donor molecule (DETA/NO) inhibited the tumor promoter-induced transformation of P+ cells. iNOS inhibitors at the micromolar concentrations that blocked NO production produced no effect on the growth of P+ cells, indicating a specific effect on transformation. Treatment of P+ cells with DETA/NO at higher concentrations (>100 µM) produced an inhibition of growth in monolayer culture that was accompanied by induction of apoptosis. However, at lower concentrations (10 µM), DETA/NO significantly inhibited NF-{kappa}B-dependent transcriptional activity with little effect on growth or apoptosis. Considering that NF-{kappa}B activation is required for transformation of JB6 cells by TNF{alpha} (40), NF-{kappa}B appears to be a functionally significant target of NO when it inhibits transformation. NO also suppressed tumor phenotype of the Tx cells by a mechanism that appears to involve induction of apoptosis. This study shows that NO suppresses both tumor promotion and tumor phenotype at doses that appear to be near physiological levels.

Although an association between iNOS overexpression and tumor phenotype has been documented, a causal role for iNOS in tumor promotion and progression has not been established (reviewed in 10, 54). A noteworthy contradiction in colon carcinogenesis with ApcMin/+ mice is that iNOS deficiency in one study (20) conferred resistance and in another study (55) conferred sensitivity to intestinal polyp formation. The explanation for the discrepancy is not clear, but may involve differences in feed, in other environmental factors, or in polyp pathology including iNOS expression levels. In ApcMin/+iNOS+/+ mice, iNOS expression in adenomas compared to normal epithelium was high in the first study, but low in the second. The exact role of iNOS in intestinal as well as other disease sites remains unresolved.

Our results demonstrate that DETA/NO at 100 µM concentration in culture medium yields NO in the nanomolar range (Fig. 3). Nanomolar NO levels would be expected to have regulatory rather than cytotoxic effects (53). Although treatment with DETA/NO inhibited both TNF{alpha}- and TPA-induced transformation in a dose-dependent manner (Fig. 4), TNF{alpha}-induced transformation was much more sensitive to inhibition by DETA/NO (at 10 µM) than TPA-induced transformation (at 500 µM). In addition to suppressing transformation, NO suppressed the tumor phenotype of Tx cells, inhibiting tumor promoter-independent colony formation significantly at 100–500 µM concentrations (Fig. 5). NO is known to inhibit DNA binding of NF-{kappa}B (56) as well as to induce apoptosis (9, 57)—possible mechanisms by which it could prevent tumor promotion and progression. DETA/NO at low concentrations (10–100 µM) significantly inhibited NF-{kappa}B-, but not AP-1-dependent transcription (Fig. 7), while at higher doses (>100 µM) DETA/NO induced apoptosis that was significantly greater in the Tx than in the P+ cells (Fig. 6B). This was in agreement with decreased viable cell numbers at higher DETA/NO concentrations (Fig. 6A). Thus, NO appears to inhibit tumor promotion by inhibiting NF-{kappa}B-dependent transcription, while this mechanism combined with induction of apoptosis may play a role in suppressing tumor phenotype.

This study not only demonstrates a protective role for NO in transformation, but also provides mechanistic understanding implicating NF-{kappa}B-dependent transcription as one target of NO-mediated suppression. In JB6 P+ cells, the AP-1 and NF-{kappa}B signaling pathways are primed for activation with the redox pathways playing a significant role (reviewed in 32). Whether increased NF-{kappa}B activity is a cause or effect of the redox state is not clear. However, the differential NF-{kappa}B activity appears to contribute to the differential induction of iNOS by TNF{alpha} in P+ and P- cells (45). It is noteworthy that expression of iNOS is not constitutive in P+ cells, but requires stimulation by TNF{alpha} or coculture with stimulated macrophages (45). This may reflect the in vivo situation of tumor initiation-promotion, in which initiated epithelial cells are responsive to cytokines like TNF{alpha} released from infiltrating macrophages. Exogenously produced NO from the macrophages themselves may also have effects on epithelial cells. The response of primary epidermal cells to endogenously or exogenously produced NO has not been ascertained. While TNF{alpha} knockout mice (TNF{alpha}-/-) are resistant to skin carcinogenesis (58), the skin carcinogenesis response of iNOS knockout mice (iNOS-/-) has not been reported. A previous report on the effect of an NO donor (nitroglycerine) on two-stage mouse skin carcinogenesis showed that local application of nitroglycerine during the promotion phase delayed papillomagenesis and reduced the number of tumors (31). In the same study, NO inhibited TPA-induced reduction of glutathione and antioxidant enzymes, as well as inhibiting promotion-required events ornithine decarboxylase (ODC) activation and elevated DNA synthesis. This result is consistent with our finding that NO has a protective role during tumor promoter-induced transformation and that suppression of iNOS and NO results in increased transformation.

In conclusion, both endogenous and exogenous NO inhibit transformation of mouse epidermal JB6 cells and suppress tumor phenotype. TNF{alpha}-induced iNOS and NO production not only do not contribute to the transformation of P+ cells, but prevent transformation. NO from a donor source inhibits transformation apparently by inhibiting NF-{kappa}B-dependent transcriptional activity. NO also induces apoptosis, which may contribute to suppression of tumor phenotype. Considering the present findings and the reported observations of conflicting evidence regarding their chemopreventive role, iNOS inhibitors will require scrutiny and reconsideration as chemopreventive agents. On the other hand, NO donors appear to have a promising role in cancer chemoprevention as well as therapy.


   Acknowledgments
 
We thank Dr. Joseph A. Hrabie (Laboratory of Chemical Carcinogenesis, National Cancer Institute, Frederick, MD) for the generous gift of DETA/NO. We also thank Dr. David Wink (Radiation Biology Branch, National Cancer Institute, Bethesda, MD) for critical review of the document and Annie Rogers (Basic Research Laboratory, National Cancer Institute, Frederick, MD) for help with editing.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Present address of Cristi Stark: FDA/CBER, Mail Stop HFM-99, Suite 200N, 1401 Rockville Pike, Rockville, MD 20852-1448.

Received 6/ 6/03; revised 8/27/03; accepted 9/ 8/03.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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