Department of Medical Elementology and Toxicology, Hamdard University, Hamdard Nagar, New Delhi 110 062, India
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 13-acetate.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 initiationpromotion 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.
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 6.22x103 m1 cm1.
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 9.6x103 m1 cm1.
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 6.22x103 m1 cm1.
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 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 TrisHCl 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 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 incorporation in the epidermal DNA by ~300% of acetone-treated 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.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 protein-phospholipase 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 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.
![]() |
Notes |
---|
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|