Carcinogenesis vol.22 no.8 pp.1207–1211, 2001
Nitroglycerin: a NO donor inhibits TPA-mediated tumor promotion in murine skin
Prashant Trikha, Nidhi Sharma and M.Athar1 Department of Medical Elementology and Toxicology, Hamdard University, Hamdard Nagar, New Delhi 110 062, India 1To
whom correspondence should be addressed Email:
[email protected]
Nitroglycerin (GTN), a nitric oxide (NO) generating vasodilator has been used in the present study to assess the role of NO during tumor promotion in murine skin. Administration of GTN to 12-O tetradecanoyl phorbol 13acetate (TPA)-treated mice resulted in a dose-dependent inhibition in the level of glutathione and the activity of antioxidant enzymes by ~16–40% of acetone-treated control. We also observed that GTN application led to a significant reduction in the ornithine decarboxylase (ODC) activity and decreased the rate of [3H]thymidine incorporation into epidermal DNA when compared with the acetonetreated control (P < 0.001). Treatment of DMBA-initiated TPA-promoted mice with GTN increased the latency period, decreased the tumor incidence by 32% and there was a 2fold decrease in tumor yield (tumor/mouse) as compared with the TPA (alone)-treated group by 20 weeks. From these data, it can be concluded that NO can abrogate the toxic and tumor promoting effects of TPA and GTN can be used as a chemopreventive agent to inhibit tumorogenesis in murine skin. Introduction Nitric oxide (NO) a short lived, potent biological molecule mediates a diverse array of roles, including vasodilation, neurotransmission, iron metabolism and immune defense (1,2). Increasing evidence suggests that NO has multiple effects on many aspects of tumor biology (3). Tumor cells capable of very high levels of NO production die in vivo, while those producing or exposed to lower levels of NO and resisting NOmediated injury undergo clonal selection (4). Thus, the effects of NO on tumor cells are dependent on the concentration of NO in the tissue and are cell type specific. Several mechanisms of NO action have been proposed, some of which are mediated through the inhibition of DNA synthesis and mitochondrial respiration. Macrophage derived NO representing endogenously produced biomolecule has been shown to inactivate complex I and II of the electron transport chain, aconitase of the Krebs’s cycle and non-haeme enzyme ribonucleotide reductase (5). NO-mediated inhibition of mitochondrion respiration and DNA synthesis may be one of the mechanisms by which activated macrophages induce cytostasis in tumor cells (6) and in microorganisms in vitro (7). NO donors have been shown to inhibit cell proliferation in colon Abbreviations: DMBA, 7,12-dimethylbenz[a]anthracene; GSH, reduced glutathione; GTN, nitroglycerin; NO, nitric oxide; ODC, ornithine decarboxylase; PMS, post-mitochondrial supernatant; TPA, 12-O-tetradecanoyl phorbol 13acetate. © Oxford University Press
carcinoma cell line (Caco-2) by inhibiting the enzyme ornithine decarboxylase, which is involved in polyamine synthesis (8). An increased level of NO synthase (NOS) expression and activity has been observed in human gynecological (9), breast (10) and central nervous system (11) tumors. In case of gynecological and breast cancer, the increased expression was inversely associated with the differentiation grade of the tumor. Excessive production of NO by inducible NOS (iNOS) has been shown to contribute to progression of human colon adenoma to carcinoma in situ (12). These observations suggest that NO may also be an important mediator in tumor growth and metastasis. Nitroglycerin (GTN) and sodium nitroprusside (SNP) which yield NO after metabolic activation, are two clinically proven vasodilators used as drugs. Both GTN and SNP inhibited DNA synthesis in RACs-I vascular smooth muscle and vascular endothelial cells in vitro (13). GTN, an organic nitrate, is biotransformed to the dinitrate metabolite following two pathways (14). In the first, biotransformation is clearance-based, resulting in the formation of inorganic nitrite (NO2–) and in the second pathway, biotransformation is mechanism-based resulting in the formation of NO and/or some related species (15). The skin tumorogenesis can be broadly divided into three stages: initiation, promotion and progression. In mouse skin, the down regulation of iNOS in the epidermis has been observed during tumor promotion (16). Transfection of iNOS into metastatic melanoma cells resulted in dramatic decrease in metastasis (17). NO generation has also been shown to delay the progression of UV-B-induced tumor in mice skin (18). The phorbol type of tumor promoter viz. TPA, mezerin and non-phorbol tumor promoter may partly act by down regulating the expression of constitutive NOS in mice skin (19). In a previous study, we have shown that GTN administration could inhibit KBrO3-mediated oxidative damage and proliferative response in kidney of rats (20). These observations prompted us to investigate the role of NO on tumor development. In the present investigation, we therefore assessed the role of exogenously produced NO on tumor promotion in DMBA-initiated and TPA-promoted mice skin. To further elucidate the mechanism through which NO may alter tumor promotion response, we studied the effect of NO on DNA synthesis. Additionally, we investigated the effect of NO release on TPA-mediated inhibition in antioxidant enzymes in mouse skin. Materials and methods Chemicals and reagents Bovine serum albumin, nicotinamide dinucleotide phosphate (reduced), phenyl methylsulfonylfluoride, pyridoxal-5-phosphate, 2-mercaptoethanol, dithiothreitol, L-ornithine, glutathione reductase, 5,5⬘-dithiobis-2-nitrobenzoic acid, oxidized glutathione, reduced glutathione, 7,12-dimethylbenz[a]anthracene (DMBA), 12-O tetradecanoyl phorbol 13-acetate (TPA), GTN were procured from Sigma Chemicals Co., St. Louis, MO, USA. [14C]ornithine (sp. act. 56 mCi/mmol), [3H]thymidine (sp. act. 82 Ci/mmol) was purchased from Amer-
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Table I. Effect of GTN application on TPA-mediated cutaneous levels of GSH and activities of antioxidant enzymes Biochemical parameters
Acetone
Glutathione (mmol/g tissue) Glutathione reductase (nmol NADPH/min/mg protein) Glutathione S-transferase (nmol CDNB/min/mg protein) Glutathione peroxidase (nmol NADPH/min/mg protein) Catalase (nmol H2O2/min/mg protein)
57.03 64.80 67.70 60.28 53.25
⫾ ⫾ ⫾ ⫾ ⫾
1.02 1.12 0.77 1.32 0.72
TPA
GTN-1
41.8 ⫾ 1.14* 42.61 ⫾ 1.18* 43.72 ⫾ 0.61* 45.04 ⫾ 0.98* 26.74 ⫾ 0.52*
45.46 51.62 51.60 49.89 33.78
GTN-2
⫾ ⫾ ⫾ ⫾ ⫾
0.65** 1.4** 0.72** 1.03** 0.58**
51.05 55.84 57.27 53.68 40.25
⫾ ⫾ ⫾ ⫾ ⫾
1.24*** 1.02*** 0.73*** 1.12*** 0.63***
Data represent mean ⫾ SE of six animals. Dose regimen and treatment protocol are given in the text. *Significant (P ⬍ 0.001) when compared with the acetone-treated control. **Significant (P ⬍ 0.01) when compared with the TPA-treated group. ***Significant (P ⬍ 0.001) when compared with the TPA-treated group.
Table II. Effect of topical application of GTN on TPA-mediated induction of epidermal ODC activity and [3H]thymidine incorporation into epidermal DNA Treatment groups
Acetone TPA TPA⫹GTN-1 TPA⫹GTN-2
[3H]thymidine incorporation
ODC activity 14CO 2
pmol released min/mg protein
155.57 ⫾ 5.58 1536.37 ⫾ 28.57* 1132.69 ⫾ 6.55** 1069 ⫾ 58.1**
% Control
[3H] DPM/mg DNA
100 988 728 687
149.9 443.86 396.42 297.5
⫾ ⫾ ⫾ ⫾
2.76 4.65* 2.43** 3.89**
% Control 100 295 264 198
Data represents mean ⫾ SE of six animals. Dose regimen and treatment protocol are given in the text. *Significant (P ⬍ 0.001) when compared with the acetone-treated control. **Significant (P ⬍ 0.001) when compared with the TPA-treated group. sham Co., Buckingham, UK. Other chemicals used in this study were of high analytical grade. Animals Swiss albino mice from Central Animal House facility of Jamia Hamdard were used for this study. Animals were housed in an air-conditioned room in polypropylene cages usually in groups of six unless mentioned otherwise. They had a free access to balanced pellet diet (Lipton Ltd, Banglore, India) and water ad libitum. The animals were kept at room temperature of 22°C (⫾2°C) and were exposed to alternate cycles of 12 h light and darkness. Treatment schedule for biochemical estimation Four groups of six animals each were used. Group I received a single topical application of 200 ml of acetone. Group II received a single application of 5 nmol TPA/200 ml of acetone. Groups III and IV received a single application of GTN at dose level of 100 mg (dose 1) and 200 mg/200 ml acetone (dose 2), respectively, 30 min after treatment of 5 nmol TPA/200 ml acetone. Twelve hours after TPA treatment, animals were killed and their dorsal skin was removed within a time of 30 min. The skin samples were immediately processed for various biochemical estimations. The treatment schedule for ornithine decarboxylase (ODC) was the same as described above except that all animals were killed 6 h after TPA treatment. For studying [3H]thymidine incorporation into epidermal DNA, animal treatment protocol and dose regimen were the same as described above. However, at the 18 h time point, animals of all groups were injected with 20 mCi [3H]thymidine/200 ml saline per mouse intraperitoneally. One hour after [3H]thymidine injection, animals were killed by cervical dislocation. Treatment of animals and tumor studies Experiments were carried out using the two-stage initiation–promotion protocol of tumorogenesis. Animals, which were at the resting phase of the hair cycle, were divided into four groups of 20 animals each. All the animals were initiated with 40 mg DMBA/200 ml acetone/mouse under subdued light. A week after initiation, promotion was started by the twice-weekly application of TPA (5 nmol/mouse) for 20 weeks. Group I animals received topical application of 200 ml acetone twice a week for 20 weeks and served as control. Group II animals were treated with 5 nmol TPA twice a week as described earlier. Groups III and IV received topical application of 100 and 200 mg of GTN twice a week, respectively, 30 min after TPA application. The number of papillomas was counted weekly. The data are expressed as percent of mice with papilloma and the number of papilloma per mouse are plotted as a function of weeks on test.
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Biochemical estimations Tissue preparation The animals were killed at the desired time period by cervical dislocation. The animals were immediately dissected to remove their skin, which was washed in ice-cold saline (0.85%) and cleaned free of extraneous material. For biochemical studies, a known amount of tissue was homogenized in a polytron homogenizer. The homogenate obtained was centrifuged at 10 500 g for 20 min to obtain post-mitochondrial supernatant (PMS). A portion of the PMS was centrifuged in an ultracentrifuge at 105 000 g for 60 min to obtain the cytosol. Reduced glutathione Reduced glutathione was estimated by the method of Jollow et al. (21). The absorbance was recorded immediately at 412 nm on a spectrophotometer (Milton Roy Model 21-D, Pittsford, New York). Glutathione reductase Glutathione reductase activity was assayed by the method of Carlberg and Mannervick (22), as modified by Mohandas et al. (23). The enzyme activity was quantified at 340 nm and was calculated as nmol NADPH oxidized/min/ mg protein using the molar extinction coefficient of 6.22⫻103 m–1 cm–1. Glutathione S-transferase Glutathione S-transferase activity was assayed by the method of Habig et al. (24) as described by Athar et al. (25). Change in absorbance was recorded at 340 nm and the enzyme activity was calculated as nmol CDNB conjugates/ min/mg protein using the molar extinction coefficient of 9.6⫻103 m–1 cm–1. Glutathione peroxidase Glutathione peroxidase activity was assayed by the method of Mohandas et al. (23). The enzyme activity was quantified at 340 nm and was calculated as nmol NADPH oxidized/min/mg protein using the molar extinction coefficient of 6.22⫻103 m–1 cm–1. Catalase activity Catalase activity was assayed by the method of Claiborne (26). Change in absorbance was recorded at 240 nm. Catalase activity was calculated in terms of nmol H2O2/min/mg protein. Nitrite estimation The amount of NO released by GTN was measured as its stable oxidative metabolite, nitrite (NO2–). Briefly, 2 ml of PMS was incubated at 37°C with 100 and 200 mg of GTN, respectively, for 30 min. PMS was mixed with an equal volume of Greiss Reagent (1:1) and allowed to stand at room temperature
GTN inhibits tumor promotion in skin for 10 min. The absorbance at 550 nm was measured and the nitrite concentration was determined using a standard curve, which was drawn using sodium nitrite as standard. Assay for ODC activity ODC activity was determined by using 0.4 ml of cytosol by the method of O’Brien et al. (27) as described by Athar et al. (25) by measuring the release of 14CO2 from D [14C]ornithine. Skin samples were homogenized in Tris– HCl buffer (pH 7.4, 50 mM) containing EDTA (1.0 mM), pyridoxal phosphate (1.0 mM), PMSF (1.0 mM), 2-mercaptoethanol (1.0 mM), dithiotheriol (0.1 mM) and Tween 80 (0.1%) at 4°C. In brief, reaction mixture consisted of 400 ml of cytosol and 0.91 ml of cofactor mixture containing EDTA (0.4 mM), pyridoxal phosphate (0.32 mM), PMSF (1.0 mM), 2-mercaptoethanol (1 mM), dithiotheriol (4.0 mM), ornithine (0.4 mM), Brij 32 (0.02%) and [14C]ornithine (0.05 mCi) in a total volume of 0.495 ml. The tubes were immediately covered with a rubber cork containing ethanolamine and methoxyethanol mixture (2:1) in the central well. After 1 h the incubation reaction was stopped by the injection of 1 ml citric acid (2.0 M) along the walls of the tube. Finally the contents of the central well were transferred to a vial containing 2.0 ml ethanol and 5 ml scintillation fluid. The amount of radioactivity was determined using a liquid scintillation counter (LKB-Wallace-1410, Pharmacia Biotech, Finland). The ODC activity was expressed as pmol 14CO2 released/h/ mg protein. Quantitation of epidermal DNA synthesis The incorporation of [3H]thymidine in epidermal DNA was measured by the method of Smart et al. (28) as described by Giri et al. (29). The skin was cleaned and 10% homogenate was prepared in cold water. The precipitate was washed with ice cold TCA (5%) and incubated with cold perchloric acid (10% PCA) overnight at 4°C. After centrifugation the precipitate was washed with cold PCA (5%). The precipitate was dissolved in warm PCA (10%) followed by incubation in a boiling water bath for 30 min and then filtered through Whatman 50 paper. The filtrate was counted for [3H]thymidine. For counting in the sample, a fixed volume of this solution was added to the scintillation vial containing 5 ml of scintillation fluid and counted in the scintillation counter (LKB-Wallace-1410). The amount of [3H]thymidine incorporation was expressed as [3H] d.p.m./mg DNA. Estimation of protein Protein was estimated by the method of Lowry et al. (30). Statistical analysis The level of significance between different groups was analyzed by Dunett’s t-test after the application of analysis of variance (ANOVA).
Results The effect of GTN administration on the TPA-mediated oxidative stress in the murine skin, was determined in terms of the levels of antioxidant peptide, GSH and the levels of all the major cutaneous antioxidant enzyme activities. As shown in Table I, TPA-alone treatment resulted in a significant inhibition in level of cutaneous glutathione, and in the activities of antioxidant enzymes namely glutathione reductase, glutathione S-transferase, glutathione peroxidase and catalase by 28, 34, 36, 25 and 49%, respectively, compared with the acetonetreated control. GTN treatment led to a dose-dependent attenuation in the level of glutathione and the activities of antioxidant enzymes by 11–20% at dose 1 and 16–40% at dose 2. The maximum amount of nitrite detected following incubation of GTN with PMS for 45 min yielded 26 and 52 mM/g tissues, respectively, at a dose of 100 and 200 mg GTN, respectively. The effect of GTN treatment on TPA-mediated induction of ODC activity and [3H]thymidine incorporation in the epidermal DNA are summarized in Table II. TPA treatment induced the activity of ODC by 990% as compared with acetone-treated control, but GTN application, led to a significant dose-dependent reduction in TPA-mediated induction of ODC by 728% at dose 1 and 687% at dose 2, respectively, compared with acetone-treated control. Similarly, a dose-dependent reduction in [3H]thymidine incorporation was observed following GTN treatment. TPA treatment resulted in increased [3H]thymidine
Fig. 1. Effect of GTN application on TPA-mediated tumor promotion in DMBA-initiated mice skin. (a) Percentage incidence of tumors plotted as a function of weeks on test. (b) Number of tumor/mice plotted as a function of number of weeks on test. Each value represents the mean incidence data of 20 animals. Dose regimen and treatment protocol are given in the text. Each value represents the mean number of tumor per mouse.
incorporation in the epidermal DNA by ~300% of acetonetreated animals, whereas in GTN-treated animals it was 265% at dose 1 and 200% at dose 2, respectively, as compared with acetone-treated (control) group. The effect of GTN treatment on DMBA-initiated TPApromoted animals is shown in Figure 1. In GTN-treated animals the latency period was slightly increased by 1 week when compared with the TPA-treated control (6 weeks in case of DMBA-initiated TPA-promoted group and 7 weeks at dose 2, in DMBA-initiated TPA-promoted and GTN-treated group). The tumor incidence was also reduced. Treatment of animals with GTN (200 mg/animal) resulted in tumor incidence of 5% at the end of 8 weeks compared with 40% incidence in DMBAinitiated TPA-promoted group by this time. In TPA-treated group the tumor incidence was 100% at end of 20 weeks, whereas it was 78 and 68% in TPA-promoted and GTN-treated group at dose 1 and dose 2, respectively, at the end of 20 weeks. GTN treatment led to a dose-dependent decrease in tumor yield. By week 15 the tumor incidence in DMBA-initiated TPA-promoted animals was 5.25 versus 2.8 in GTN-treated (dose 2) animals. Similarly, the tumor yield was 4.45 and 3.4 tumor/mouse at dose 1 and dose 2, respectively, in the GTNtreated group, whereas it was 6.75 in the DMBA-initiated TPA-treated group by the end of 20 weeks. 1209
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Discussion Tumor promotion is known to involve oxidative stress at least during the early stages. Therefore, most tumor promoters have been shown to act through the elaboration of pro-oxidant response (31,32). TPA, a phorbol type tumor promoter has been shown to induce oxidative stress in murine skin by the generation of free radicals (33). TPA treatment resulted in the inhibition of the activity of all the major cutaneous antioxidant enzymes. Application of GTN- to TPA-treated mice ameliorated TPA-mediated oxidative stress, this is evident by the attenuation in the level of glutathione and the increase in the activities of all the major antioxidant enzymes. The NO liberated from GTN is itself a free radical and can quench other free radicals particularly O2–√ (superoxide anion), and this may contribute to its protective effect (34). The excessive NO production and oxidant generation simultaneously leads to the formation of non-toxic metabolites like nitrite and nitrates. This prevents the action of reactive oxygen species on DNA, thereby providing a mechanism of protecting against DNA damage. Lipoxygenase, which releases clastogenic agents and endoperoxides, is induced by TPA, NO has been shown to inhibit lipoxygenase activity, which provides an additional mechanism for the antitumorogenic action of NO (35). During tumor promotion in mouse skin an inverse correlation between NOS generation and tumor promotion has been observed, suggesting that NO plays a crucial role in cellular proliferation (16). When the kinetic trend of TPA on constitutive NOS activity was compared with the time course response of TPA on ODC activity, an inverse correlation was seen as the ODC activity increased; there was a concurrent decrease (down regulation) in NOS activity. This suggests a common pathway in TPA-mediated tumor promotion in skin (19). NO has also been shown to inhibit certain key enzymes involved in DNA synthesis like ribonucleotide reducatse (36) and ornithine decarboxylase (8). Induction of ODC activity and [3H]thymidine incorporation into epidermal DNA are extensively used biochemical markers to evaluate TPA-mediated tumor promotion in murine skin (37). Based on this information, we have assessed the effect of GTN on TPA-mediated induction of biochemical responses of tumor promotion. The application of GTN to TPA-treated animals led to a significant diminution in the activity of ODC and decreased rate of [3H]thymidine incorporation into the epidermal DNA, which further provides evidence that NO inhibits cellular proliferation. Administration of NO donors during TPA-mediated tumor promotion resulted in the decrease in tumor incidence, which suggests that NO may regulate tumor growth during the promotion stage of carcinogenesis. NO has been shown to participate in the signal transduction pathway via cGMP activation (38). Several NO donors have been shown to activate cGMP level in vitro and in CAM models (39,40). The molecular mechanisms leading to tumor promotion by phorbol esters involve phospholipase C and PKC activation. It is believed that cGMP may inhibit PKC-dependent pathways by interfering with the processes downstream from receptor G proteinphospholipase C effector system (38). PKC down regulates NOS through phosphorylation, but the precise mechanism by which elevated cGMP inhibits cellular proliferation is unclear. Some studies, however, show that cGMP induces hyperpolarization of cell membrane, which inhibits Ca2⫹ inflow (41). The decrease in intracellular Ca2⫹ inhibits the activity of PKC 1210
leading to the inhibition of PKC-dependent proliferation (42). This could be one of the mechanism by which NO exerts its inhibitory role during tumor promotion. Another important mechanism by which NO donors may act to reduce tumorogenesis is the enhancement of apoptosis. Exposure of cells to NO also leads to apoptosis. DNA damage caused by NO exposure leads to p53 accumulation, which causes growth arrest in the G1 phase of cell cycle and ultimately leads to apoptosis (43). In addition, NO may also influence the expressions of genes related to tumorogenesis such as p53, Bcl-2 and Fas (44,45). NO can selectively inhibit the expression of matrix metalloprotease, which play an important role in angiogenesis and metastasis (46). The results of our study demonstrate that exogenously produced NO may play a critical role in protecting against the deleterious effect of superoxide anions generated by TPA. In addition NO can inhibit cellular proliferation by down regulating the activity of ODC and the rate of thymidine incorporation into epidermal DNA during TPA-mediated tumor promotion. Acknowledgements The authors are grateful to Mr Shiraz Hussain, Vice-chancellor, Jamia Hamdard for providing necessary facilities. P.T. and N.S. are grateful to the Lady Tata Memorial Trust, Mumbai, India for providing fellowship.
References 1. Nathan,C. and Xie,Q.W. (1994) Nitric oxide synthases: roles, tolls and controls. Cell, 78, 915–918. 2. Cui,S., Reichner,J.S., Mateo,R.B. and Albina,J.E. (1994) Activated murine macrophages induce apoptosis in tumor cells through nitric oxide dependent or independent mechanisms. Cancer Res., 54, 2462–2467. 3. Shi,Q., Huang,S., Jiang,W., Kutach,L.S., Ananthaswamy,H.N. and Xie,K. (1999) Direct correlation between nitric oxide synthase II inducibility and metastatic ability of UV-2237 murine fibrosarcoma cells carrying mutant p53. Cancer Res., 59, 2072–2075. 4. Xie,K. and Fidler,I.J. (1998) Therapy of cancer metastasis by activation of the inducible nitric oxide synthase. Cancer Metastasis Rev., 17, 55–75. 5. Drapier,J.C. and Hibbs,J.B. (1986) Murine cytotoxic activated macrophages inhibit aconitase in tumor cells. Inhibition involves the iron-sulfur prosthetic group and is reversible. J. Clin. Invest., 78, 790–797. 6. Stuehr,D.J. and Nathan,C.F. (1989) A macrophage product responsible for cytostasis and repiratory inhibition in tumor target cells. J. Exp. Med., 169, 1543–1555. 7. Granger,J.E., Hibbs,J.B., Perfect,J.R. and Durack,D.T. (1988) Specific amino acid (L-arginine) requirement for the microbiostatic activity of murine macrophages. J. Clin. Invest., 81, 1129–1136. 8. Bayer,P.M., Fukuto,J.M., Buga,G.M., Pegg,A.E. and Ignarro,L.J. (1999) Nitric oxide inhibits ornithine decarboxylase by S-nitrsosylation. Biochem. Biophys. Res. Commun., 262, 355–358. 9. Thomsen,L.L., Lawton,F.G., Knowles,R.G., Beesley,J.E., RiverosMoreno,V. and Moncada,S. (1994) Nitric oxide synthase activity in human gynecological cancer. Cancer Res., 54, 1352–1354. 10. Thomsen,L.L., Miles,D.W., Happerfield,L., Bobrow,L.G., Knowles,R.G. and Moncada,S. (1995) Nitric oxide synthase activity in human breast cancer. Br. J. Cancer, 72, 41–44. 11. Cobbs,C.S., Brenman,J.E., Aldape,K.D., Bredt,D.S. and Israel,M.A. (1995) Expression of nitric oxide synthase in human central nervous system tumors. Cancer Res., 55, 727–730. 12. Ambs,S., Merriam,W.G., Bennett,W.P., Felley-Bosco,E., Ogunfusika,M.O., Oser,S.M., Klein,S., Shields,P.G., Billiar,T.R. and Harris,C.C. (1998) Frequent nitric oxide synthase-2 expression in human colon adenomas: implication for tumor angiogenesis and colon progression. Cancer Res., 58, 334–341. 13. Nakaki,T., Nakayama,M. and Kato,R. (1990) Inhibition by nitric oxide and nitric oxide-producing vasodilators of DNA synthesis in vascular smooth muscle cells. Eur. J. Pharmacol., 189, 347–353. 14. Knowles,R.G., Palacios,M., Palmer,R.M.J. and Moncada,S. (1989) Formation of nitric oxide from L-arginine in the central nervous system. Proc. Natl Acad. Sci. USA, 86, 5159–5162.
GTN inhibits tumor promotion in skin 15. Adami,A., Crivellente,F., Prati,A.C., Cavalieri,E., Cuzzolin,L., Tommasi,M., Suzuki,H. and Benoni,G. (1998) Biotransformation and cytotoxic properties of NO-donors on MCF7 and U251 cell lines. Life Sci., 63, 2097–2105. 16. Robertson,F.M., Long,B.W., Tober,K.L., Mary,S.R. and Tatiana,M.O. (1996) Gene expression and cellular sources of inducible nitric oxide synthase during tumor promotion. Carcinogenesis, 17, 2053–2061. 17. Xie,K., Huang,S., Dong,Z., Gutman,M. and Fidler,I.J. (1995) Direct correlation between expression of endogenous inducible nitric oxide synthase and regression of M5076 reticulum cell sarcoma hepatic metastasis in mice treated with liposome containing lipopeptide CGP 31362. Cancer Res., 55, 3123–3131. 18. Yim,C.Y., Bastian,N.R., Smith,J.C., Hibbs,J.B. and Samlowski,W.E. (1993) Macrophage nitric oxide synthesis delays progression of ultraviolet lightinduced murine skin cancers. Cancer Res., 53, 5507–5511. 19. Ahmad,N., Srivastava,R.C., Agarwal,R. and Mukhtar,H. (1997) Nitric oxide synthase and skin tumor promotion. Biochem. Biophys. Res. Commun., 232, 328–331. 20. Rehman,A., Saluddin,A., Khan,N., Sultana,S. and Athar,M. (1999) Glyceryl trinitrate, a nitric oxide donor, suppresses renal oxidant damage caused by potassium bromate. Redox Report, 4, 263–269. 21. Jollow,D.J., Mitchell,J.R., Zampagilone,N. and Gilete,J.R. (1974) Bromobenzene-induced liver necrosis: protective role of glutathione and evidence for 3,4-bromobenzene oxide as the hepatotoxic intermediate. Pharmacology, 11, 151–169. 22. Carlberg,I. and Mannerviek,B. (1975) Glutathione reductase levels in rat brain. J. Biol. Chem., 250, 5475–5480. 23. Mohandas,J., Marshsall,J.J., Duggins,G.G., Horvath,J.S. and Tiller,D. (1984) Differential distribution of glutathione and glutathione related enzymes in rabbit kidney. Possible implications in analgesic neuropathy. Cancer Res., 44, 5086–5091. 24. Habig,W.H., Pabst,M.J. and Jokoby,W.B. (1974) Glutathione S-transferase. The first enzymatic step in mercapturic acid formation. J. Biol. Chem., 249, 7130–7139. 25. Athar,M., Khan,W.A. and Mukhtar,H. (1989) Effect of dietary tannic acid on epidermal, lung and forestomach polycyclic aromatic hydrocarbon metabolism and tumorogenicity in sencar mice. Cancer Res., 49, 5784– 5788. 26. Claiborne,A. (1985) Catalase activity. In Greenwald,R.A. (ed.) CRC Handbook of Methods For Oxygen Radical Research. CRC Press, Boca Raton, FL, pp. 283–284. 27. O’Brien,T.G., Simsiman,R.C. and Boutwell,R.K. (1975) Induction of the polyamine biosynthetic enzymes in mouse epidermis by tumor-promoting agents. Cancer Res., 35, 1662–1670. 28. Smart,R.C., Huang,M.T. and Couney,A.H. (1986) Sn 1,2-diacylglycerols mimic the effects of TPA in vivo by inducing biochemical changes associated with tumor promotion in mouse epidermis. Carcinogenesis, 7, 1865–1870. 29. Giri,U., Sharma,S.D., Abdulla,M. and Athar,M. (1996) Porphyrin-mediated photosensitization has a weak tumor promoting activity in mouse skin: possible role of in situ generated reactive oxygen species. Carcinogenesis, 17, 2023–2028.
30. Lowry,O.H., Rosenbrough,N.J., Farr,A.L. and Randall,R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265–275. 31. Cerutti,P.A. (1994). Oxy-radicals and cancer. Lancet, 344, 862–863. 32. Kensler,T.W. and Taffe,B.G. (1989) Role of free radicals in tumor promotion and progression. Prog. Clin. Biol. Res., 298, 233–248. 33. Giri,U., Iqbal,M., Agarwal,M.K., Khan,S. and Athar,M. (1999) Augmentation of 12-O-tetradecanoyl 13-phorbol acetate-mediated tumor promoting response by the porphyrin photosensitization of 7,12-dimethyl benz[a]anthracene-initiated murine skin: role of in situ generated reactive oxygen species. Cancer Lett., 135, 53–60. 34. Parker-Botelho,L., Cantor,T.H., Ho,E.H., Lumma,W.C. and Rubanyi,G.M. (1989) Nitric oxide inactivates superoxide radicals produced by human leukocytes. Circulation, 80, 115. 35. Maccarrone,M., Corasaniti,M.T., Guerrieri,P., Nistico,G. and Agro,A.F. (1996) Nitric oxide-donor compounds inhibit lipoxygenase activity. Biochem. Biophys. Res. Commun., 219, 128–133. 36. Schmidt,H.H.W., Pollock,J.S., Nakane,M., Forstermann,U. and Murad,F. (1992) Calcium/calmodulin regulated nitric oxide synthase. Cell, 13, 427–434. 37. O’Brien,T.G. (1976) The induction of ornithine decarboxylase an early, possible obligatory event in mouse skin. Carcinogenesis, 36, 2644–2653. 38. Doni,M.G., Deana,R., Padoin,E., Ruzzene,M. and Alexandre,A. (1991) Platelet activation by diacylglycerol or ionomycin is inhibited by nitroprusside. Biochem. Biophys. Acta, 1094, 323–329. 39. Pipili-Synetos,E., Sakkoula,E., Haralabopoulos,G., Andriopoulou,P., Peristen,P. and Maragoudakis,M.E. (1994) Evidence that nitric oxide is an endogenous antiangiogenic mediator. Br. J. Pharmacol., 111, 894–902. 40. Pipili-Synetos,E., Papageorgiou,A., Sakkoula,E., Sotiropoulou,G., Fotsis,T., Karakiulakis,G. and Maragoudakis,M.E. (1995) Inhibition of angiogenesis, tumor growth and metastasis by the NO-releasing vasodilators, isosorbide mononitrate and dinitrate. Br. J. Pharmacol., 116, 1829–1834. 41. Yang,W., Ando,J., Korenga,R., Toyo,T. and Kamiya,A. (1994) Exogenous nitric oxide inhibits proliferation of cultured vascular endothelial cells. Biochem. Biophys. Res. Commun., 203, 1160–1167. 42. DiGiovanni,J. (1992) Multistage carcinogenesis in mouse skin. Pharmac. Ther., 54, 63–128. 43. Kastan,M.B., Canman,C.E. and Leonard,C.J. (1995) p53, cell cycle control and apoptosis: implication for cancer. Cancer Met. Rev., 14, 3–15. 44. Nishio,E., Fukushima,K., Shiozaki,M. and Watanabe,Y. (1996) Nitric oxide donor SNAP induces apoptosis in smooth muscle cells through cGMPindependent mechanism. Biochem. Biophys. Res. Commun., 221, 163–168. 45. Brune,B., Mohr,S. and Messmer,U.K. (1996) Protein thiol modification and apoptotic cell death as cGMP independent nitric oxide (NO) signaling pathways. Rev. Physiol. Biochem. Pharmacol., 127, 1–30. 46. Xie,K. and Fidler,I.J. (1997) Decreased matrix metalloproteinase- (MMP2) expression correlates with suppression of tumorogenicity and metastasis of K-1735 murine melanoma cells transfected with the inducible nitric oxide synthase (iNOS) gene. Proc. Am. Assoc. Cancer. Res., 38, 1113. Received November 22, 2000; revised March 23, 2001; accepted April 18, 2001
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