3-nitrotyrosine and Colonic-Neoplasms

The cancer preventive activity of vitamin E has been extensively discussed, but the activities of specific forms of tocopherols have not received sufficient attention. Herein, we compared the activities of δ-tocopherol (δ-T), γ-T, and α-T in a colon carcinogenesis model. Male F344 rats, seven weeks old, were given two weekly subcutaneous injections of azoxymethane (AOM) each at a dose of 15 mg/kg body weight. Starting 1 week before the AOM injection, the animals were maintained on a modified AIN76A diet, or the same diet containing 0.2% of δ-T, γ-T, α-T, or a γ-T-rich mixture of tocopherols (γ-TmT), until the termination of the experiment at 8 weeks after the second AOM injection. δ-T treatment showed the strongest inhibitory effect, decreasing the numbers of aberrant crypt foci by 62%. γ-T and γ-TmT were also effective, but α-T was not. Immunohistochemical analysis showed that δ-T and γ-T treatments reduced the levels of 4-hydroxynonenal and nitrotyrosine and the expression of cyclin D1 in the colon, preserved the expression of PPAR-γ, and decreased the serum levels of prostaglandin E2 and 8-isoprostane. Supplementation with 0.2% δ-T, γ-T, or α-T increased the respective levels of tocopherols and their side-chain degradation metabolites in the serum and colon tissues. Rather high concentrations of δ-T and γ-T and their metabolites were found in colon tissues. Our study provides the first evidence for the much higher cancer preventive activity of δ-T and γ-T than α-T in a chemically induced colon carcinogenesis model. It further suggests that δ-T is more effective than γ-T.

Topics: Aldehydes; alpha-Tocopherol; Animals; Anticarcinogenic Agents; Azoxymethane; Colonic Neoplasms; Cyclin D1; Dinoprost; Dinoprostone; gamma-Tocopherol; Immunohistochemistry; Male; Models, Chemical; Rats; Rats, Inbred F344; Tocopherols; Tyrosine

Chronic inflammation increases the risk of development of several types of malignancies including colon cancer. It also represents a paradigm for the connection between inflammation and cancer in terms of epidemiology and mechanistic studies in preclinical models. A key component of inflammation promoting cancer is the transcription factor NF-κB, which is known to play a critical role in the regulation of the inducible nitric oxide synthase (iNOS) gene. iNOS is an enzyme dominantly expressed during inflammatory reactions. Although synthesis of high amounts of nitric oxide (NO) by iNOS has been demonstrated in pathophysiological processes, such as acute or chronic inflammation and tumorigenesis, the role of iNOS activity in these diseases is still not well understood. Analysis of human biopsies of colitis and colon cancer using immunohistochemistry revealed elevated iNOS protein expression levels, which were strongly paralleled by increased expression of nitrotyrosine suggesting that iNOS has been highly activated in these tissues. These results were corroborated in an in vitro study showing the presence of high iNOS levels in a colon cancer cell line (HT-29) following inflammatory stimuli (TNF-α, peroxynitrite). In addition, the involvement of metastatic processes in the colon biopsies was assessed by means of in situ zymography of MMP activation. MMP 2 (gelatinase A) activation was higher in histopathological sections of colitis and cancer compared to controls. Overall, these data strengthen the findings that in inflammation and colon cancer in humans, iNOS expression and tyrosine nitration may be an indicator of cancer development and progression.

Topics: Colitis; Colonic Neoplasms; HT29 Cells; Humans; Immunohistochemistry; NF-kappa B; Nitric Oxide Synthase Type II; Peroxynitrous Acid; Real-Time Polymerase Chain Reaction; Tumor Cells, Cultured; Tyrosine

We investigated the effects of a gamma-tocopherol-rich mixture of tocopherols (gamma-TmT, containing 57% gamma-T, 24% delta-T, and 13% alpha-T) on colon carcinogenesis in azoxymethane (AOM)/dextran sulfate sodium (DSS)-treated mice. In experiment 1, 6-week-old male CF-1 mice were given a dose of AOM (10 mg/kg body weight, i.p.), and 1 week later, 1.5% DSS in drinking water for 1 week. The mice were maintained on either a gamma-TmT (0.3%)-enriched or a standard AIN93M diet, starting 1 week before the AOM injection, until the termination of experiment. In the AOM/DSS-treated mice, dietary gamma-TmT treatment resulted in a significantly lower colon inflammation index (52% of the control) on day 7 and number of colon adenomas (9% of the control) on week 7. gamma-TmT treatment also resulted in higher apoptotic index in adenomas, lower prostaglandin E2, leukotriene B4, and nitrotyrosine levels in the colon, and lower prostaglandin E2, leukotriene B4, and 8-isoprostane levels in the plasma on week 7. Some of the decreases were observed even on day 7. In experiment 2 with AOM/DSS- treated mice sacrificed on week 21, dietary 0.17% or 0.3% gamma-TmT treatment, starting 1 week before the AOM injection, significantly inhibited adenocarcinoma and adenoma formation in the colon (to 17-33% of the control). Dietary 0.3% gamma-TmT that was initiated after DSS treatment also exhibited a similar inhibitory activity. The present study showed that gamma-TmT effectively inhibited colon carcinogenesis in AOM/DSS-treated mice, and the inhibition may be due to the apoptosis-inducing, anti-inflammatory, antioxidative, and reactive nitrogen species-trapping activities of tocopherols.

Topics: Adenocarcinoma; Adenoma; Animals; Antioxidants; Apoptosis; Azoxymethane; Carcinogens; Cell Transformation, Neoplastic; Cocarcinogenesis; Colon; Colonic Neoplasms; Dextran Sulfate; Dinoprost; Dinoprostone; Dose-Response Relationship, Drug; gamma-Tocopherol; Inflammation; Leukotriene B4; Male; Mice; Tyrosine

Methylamine dichloramine (CH(3)NCl(2)) produced by neutrophils may promote colon tumors and colitis via architectural and oxidative changes in crypts, which are secretory granulae composed of goblet cells located in the colorectal mucosal layer. We investigated whether CH(3)NCl(2), in comparison with the other reactive oxygen species (ROS) such as H(2)O(2) and HOCl, derived from primed neutrophils in inflammatory sites in the large intestine, is a biogenic factor for the induction of colorectal disease in mice.. Male ICR-strain mice were administered each oxidant (0.5-0.7 micromol/mouse) by enema under anesthesia. The colorectal tissues were evaluated by histopathological and immunohistochemical analyses. Hemolysis and hemoglobin oxidation by the methylamine chloramines and HOCl were examined by adding them (50-400 microM) to a sheep erythrocyte suspension (1x10(8) cells/ml) and its lysate at pH 7 and 37 degrees C.. CH(3)NCl(2) oxidized erythrocyte hemoglobin more effectively than HOCl, indicating it has high cell permeability and selective oxidation ability. CH(3)NCl(2) mainly induced atrophy of crypts at 6 h after administration, while the other ROS tested did not. Furthermore, 4-hydroxy-2-nonenal (4-HNE) showed positive immunostains throughout the mucosal layer, including around the basal regions of atrophied crypts, only with CH(3)NCl(2), while positive immunostains were observed for 3-nitrotyrosine (3-NT) in the atrophied crypts and their surrounding lamina propria in the mucosal layer.. The results suggest that CH(3)NCl(2)derived from primed neutrophils may play the most important role in promoting the development of colon tumor formation and colitis by oxidative stress through its high degree of cell permeability.

Topics: Aldehydes; Animals; Chloramines; Colon; Colonic Neoplasms; Hemoglobins; Hemolysis; Hydrogen Peroxide; Hypochlorous Acid; Immunohistochemistry; Inflammatory Bowel Diseases; Intestinal Mucosa; Male; Mice; Mice, Inbred ICR; Neutrophil Activation; Oxidation-Reduction; Sheep; Tyrosine

In Japan, rice vinegar that has been matured and fermented for years in earthenware jars is considered a health food with anticolon cancer action. It is divided into the liquid component (Kurozu) and the sediment (Kurozu moromimatsu), which contains large amounts of organic materials and minerals. The effect of Kurozu moromimatsu (Kurozu-M) on cancer has not yet been examined. In this study, we examined the activity of Kurozu-M on colon cancer and investigated the mechanisms involved, focusing on active oxygen generation, apoptosis, and metalloproteinases (MMPs).. We used Lovo cells transplanted into nude mice as an experimental model. We measured the tumor volume and MMP levels and conducted hematoxylin-eosin staining (for polymorphonuclear leukocytes), terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining (for apoptosis), and immunostaining for nitrotyrosine (a marker of active oxygen generation) in control, Kurozu-treated, and Kurozu-M--treated groups.. The tumor volume was the same in the control group (231 +/- 36 mm(3)) and Kurozu group (238 +/- 52 mm(3)), but was significantly reduced in the Kurozu-M group (152 +/- 28 mm(3), P < 0.001 versus control). Apoptosis of tumor cells and accumulation of polymorphonuclear leukocytes were not observed. Nitrotyrosine production, total MMP levels, and MMP activation were significantly reduced in the Kurozu-M group.. The administration of Kurozu-M prolonged the lifespan of cancer cell-transplanted mice, inhibited tumor progression, and reduced nitrotyrosine production and MMP activation, but did not induce apoptosis.

Topics: Animals; Antineoplastic Agents; Apoptosis; Colonic Neoplasms; Female; Fermentation; Metalloproteases; Mice; Mice, Nude; Neoplasm Transplantation; Oryza; Phytotherapy; Plant Extracts; Random Allocation; Specific Pathogen-Free Organisms; Tyrosine

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors are known to modulate carcinogenesis. In this study, we investigated whether a lipophilic HMG-CoA reductase inhibitor pitavastatin suppresses inflammation-related mouse colon carcinogenesis. Male CD-1 (ICR) mice were initiated with a single intraperitoneal injection of azoxymethane (AOM, 10 mg/kg body weight) and promoted by 2% (w/v) dextran sodium sulfate (DSS) in drinking water for 7 days. The experimental diets containing pitavastatin at 2 dose levels (1 and 10 ppm) were fed to male CD-1 (ICR) mice for 17 weeks, staring 1 week after the cessation of DSS exposure. The effects of dietary pitavastatin on colonic tumor development were assessed at Weeks 5, 10 and 20. Feeding with pitavastatin at both doses significantly inhibited the multiplicity of colonic adenocarcinoma at Week 20. Furthermore, the treatment significantly lowered the positive rates of proliferating cell nuclear antigen and increased the apoptotic index in the colonic epithelial malignancies. The treatment also reduced nitrotyrosine-positivity in the colonic mucosa. Our findings thus show that pitavastatin is effective in inhibiting colitis-related colon carcinogenesis through modulation of mucosal inflammation, oxidative/nitrosative stress, and cell proliferation.

Topics: Animals; Body Weight; Cholesterol; Colonic Neoplasms; DNA, Single-Stranded; Immunohistochemistry; Inflammation; Male; Mice; Molecular Structure; Organ Size; Proliferating Cell Nuclear Antigen; Quinolines; Time Factors; Triglycerides; Tyrosine

Arginine is catabolized by NOS2 and other nitric oxide synthases to form nitric oxide. We evaluated the roles of dietary arginine and Nos2 in Apc-dependent intestinal tumorigenesis in Min mice with and without a functional Nos2 gene. NOS2 protein was expressed only in intestinal tissues of Apc(Min/+) Nos2+/+ mice. NOS3 expression was higher in intestinal tissues of mice lacking Nos2, mainly in the small intestine. When diet was supplemented with arginine (0.2% and 2% in drinking water), lack of Nos2 results in decreased tumorigenesis in both small intestine and colon. In Nos2 knockout mice, supplemental arginine (up to 2%) caused a decrease in small intestinal tumor number and size. The arginine-dependent decrease was associated with an increase in nitrotyrosine formation and apoptosis in the region of intestinal stem cells. Mice expressing Nos2 did not show these changes. These mice did, however, show an arginine-dependent increase in colon tumor number and incidence, while no effect on apoptosis was seen. These changes were associated with increased nitrotyrosine formation in epithelial cells. Mice lacking Nos2 did not show changes in tumorigenesis or nitrotyrosine formation, while demonstrating an arginine-dependent increase in apoptosis. These data suggest that Nos2 and dietary arginine have significant effects on intestinal and colonic tumorigenesis in Min mice. In both tissues, loss of Nos2 is associated with decreased tumorigenesis when mice are supplemented with dietary arginine. In the small intestine, Nos2 prevents the arginine-induced decrease in tumor number and size, which is associated with NOS3 expression and increased apoptosis. In the colon, Nos2 is required for the arginine-induced increase in tumor number and incidence.

Topics: Adenomatous Polyposis Coli Protein; Animals; Apoptosis; Arginine; Colonic Neoplasms; Dietary Supplements; Female; Intestinal Neoplasms; Intestine, Small; Male; Mice; Mice, Knockout; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Tyrosine

Studies have suggested that diets rich in polyphenols such as flavonoids may lead to a reduced risk of gastrointestinal cancers. We demonstrate the ability of monomeric and dimeric flavanols to scavenge reactive nitrogen species derived from nitrous acid. Both epicatechin and dimer B2 (epicatechin dimer) inhibited nitrous acid-induced formation of 3-nitrotyrosine and the formation of the carcinogenic N-nitrosamine, N-nitrosodimethylamine. The reaction of monomeric and dimeric epicatechin with nitrous acid led to the formation of mono- and di-nitroso flavanols, whereas the reaction with hesperetin resulted primarily in the formation of nitrated products. Although, epicatechin was transferred across the jejunum of the small intestine yielding metabolites, its nitroso form was not absorbed. Dimer B2 but not epicatechin monomer inhibited the proliferation of, and triggered apoptosis in, Caco-2 cells. The latter was accompanied by caspase-3 activation and reductions in Akt phosphorylation, suggesting activation of apoptosis via inhibition of prosurvival signaling. Furthermore, the dinitroso derivative of dimer B2, and to a lesser extent the dinitroso-epicatechin, also induced significant toxic effects in Caco-2 cells. The inhibitory effects on cellular proliferation were paralleled by early inhibition of ERK 1/2 phosphorylation and later reductions in cyclin D1 levels, indicating modulation of cell cycle regulation in Caco-2 cells. These effects highlight multiple routes in which dietary derived flavanols may exert beneficial effects in the gastrointestinal tract.

Topics: Absorption; Animals; Apoptosis; Caco-2 Cells; Caspase 3; Caspases; Catechin; Cell Cycle; Cell Proliferation; Colonic Neoplasms; Cyclin D1; Dimethylnitrosamine; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Flavonoids; Gastrointestinal Tract; Humans; In Vitro Techniques; Mitogen-Activated Protein Kinase Kinases; Nitrosamines; Nitroso Compounds; Nitrous Acid; Phenols; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Reactive Nitrogen Species; Time Factors; Tyrosine

We previously reported a powerful tumor-promoting ability of dextran sodium sulfate (DSS) in a novel mouse model for colitis-related colon carcinogenesis initiated with azoxymethane (AOM). To determine the dose-dependent influence of DSS in our animal model, male ICR mice were given a single intraperitoneal injection of AOM (10 mg/kg body weight), followed by DSS at dose levels of 2, 1, 0.5, 0.25, and 0.1% (w/v) in drinking water for 1 week. All animals were sacrificed at week 14 and histological alterations in their colon and nitrotyrosine immunohistochemistry were examined to evaluate the nitrosative stress. In the mice which received AOM and 2% DSS, the incidences (multiplicity) of colonic tubular adenoma and adenocarcinoma were 75% (1.25+/-1.26/mouse) and 100% (2.75+/-2.22/mouse), respectively. Mice given AOM and 1% DSS had 80% incidence of adenoma (1.00+/-0.71/mouse) and 60% incidence of adenocarcinoma (1.40+/-2.07/mouse) in the colon. In a mouse treated with AOM and 0.5% DSS, only one colonic adenoma (20% incidence with 0.20+/-0.45 multiplicity) developed. Higher frequency of high-grade colonic dysplasia was noted in mice given AOM and 2% or 1% DSS when compared with mice treated with AOM and lower doses of DSS. Also, scoring of inflammation and nitrotyrosine immunoreactivity suggested that severe inflammation and nitrosation stress caused by high-doses (2% and 1%) of DSS contribute its tumor-promoting effects in mouse colon carcinogenesis initiated with a low dose of AOM. Thus, our findings indicate that a tumor-promoting effect of DSS was dose-dependent (1% or more) and the effect might occur under the condition of inflammation and nitrosation stress.

Topics: Animals; Azoxymethane; Carcinogens; Cocarcinogenesis; Colitis; Colonic Diseases; Colonic Neoplasms; Dextran Sulfate; Dose-Response Relationship, Drug; Intestinal Mucosa; Male; Mice; Mice, Inbred ICR; Tyrosine; Ulcer

It is generally assumed that inflammatory bowel disease (IBD)-related carcinogenesis occurs as a result of chronic inflammation. We previously developed a novel colitis-related mouse colon carcinogenesis model initiated with azoxymethane (AOM) and followed by dextran sodium sulfate (DSS). In the present study we investigated whether a cyclooxygenase (COX)-2 inhibitor nimesulide and ligands for peroxisome proliferator-activated receptors (PPARs), troglitazone (a PPARgamma ligand) and bezafibrate (a PPARalpha ligand) inhibit colitis-related colon carcinogenesis using our model to evaluate the efficacy of these drugs in prevention of IBD-related colon carcinogenesis.. Female CD-1 (ICR) mice were given a single intraperitoneal administration of AOM (10 mg/kg body weight) and followed by one-week oral exposure of 2% (w/v) DSS in drinking water, and then maintained on the basal diets mixed with or without nimesulide (0.04%, w/w), troglitazone (0.05%, w/w), and bezafibrate (0.05%, w/w) for 14 weeks. The inhibitory effects of dietary administration of these compounds were determined by histopathological and immunohistochemical analyses.. Feeding with nimesulide and troglitazone significantly inhibited both the incidence and multiplicity of colonic adenocarcinoma induced by AOM/DSS in mice. Bezafibrate feeding significantly reduced the incidence of colonic adenocarcinoma, but did not significantly lower the multiplicity. Feeding with nimesulide and troglitazone decreased the proliferating cell nuclear antigen (PCNA)-labeling index and expression of beta-catenin, COX-2, inducible nitric oxide synthase (iNOS) and nitrotyrosine. The treatments increased the apoptosis index in the colonic adenocarcinoma. Feeding with bezafibrate also affected these parameters except for beta-catenin expression in the colonic malignancy.. Dietary administration of nimesulide, troglitazone and bezafibrate effectively suppressed the development of colonic epithelial malignancy induced by AOM/DSS in female ICR mice. The results suggest that COX-2 inhibitor and PPAR ligands could serve as an effective agent against colitis-related colon cancer development.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Bezafibrate; Body Weight; Chromans; Colitis; Colonic Neoplasms; Cyclooxygenase 2; Cyclooxygenase Inhibitors; Female; Hypolipidemic Agents; Immunohistochemistry; Inflammation; Injections, Intraperitoneal; Ligands; Male; Mice; Mice, Inbred ICR; Nitric Oxide Synthase Type II; Organ Size; Peroxisome Proliferator-Activated Receptors; Sulfonamides; Temperature; Thiazolidinediones; Troglitazone; Tyrosine; Vasodilator Agents

Previously, we proposed a novel mouse model for colitis-related colon carcinogenesis using azoxymethane (AOM) and dextran sodium sulfate (DSS) (Cancer Sci 2003; 94: 965-73). In the current study, sequential analysis of pathological alterations during carcinogenesis in our model was conducted to establish the influence of inflammation caused by DSS on colon carcinogenesis in this model. Male ICR mice were given a single intraperitoneal injection of AOM (10 mg/kg body weight) and given 2% (w/v) DSS in the drinking water for 7 days, starting 1 week after the AOM injection. They were sequentially sacrificed at weeks 2, 3, 4, 5, 6, 9, 12, and 14 for histopathological and immunohistochemical examinations. Colonic adenomas were found in 2 (40% incidence and 0.40 +/- 0.49 multiplicity) of 5 mice at week 3 and colon carcinomas developed in 2 (40% incidence and 2.00 +/- 3.52 multiplicity) of 5 mice at week 4. Their incidence gradually increased with time and reached 100% (6.20 +/- 2.48 multiplicity) at week 6. At week 14, the multiplicity of adenocarcinoma was 9.75 +/- 2.49 (100% incidence). In addition, colonic dysplasia was noted at all time-points. The scores of colonic inflammation and nitrotyrosine immunohistochemistry were extremely high at early time-points and were well correlated. Our results suggest that combined treatment of mice with AOM and DSS generates neoplasms in the colonic mucosa via dysplastic lesions induced by nitrosative stress.

Topics: Adenocarcinoma; Animals; Azoxymethane; Carcinogens; Colitis; Colon; Colonic Neoplasms; Dextran Sulfate; Immunoenzyme Techniques; Incidence; Injections, Intraperitoneal; Intestinal Mucosa; Male; Mice; Mice, Inbred ICR; Precancerous Conditions; Tyrosine

Peroxynitrite (ONOO(-)), which is generated from nitric oxide (NO) and superoxide anion (O(2)(.-)) under pathological conditions, plays an important role in pathophysiological processes. Activation of matrix metalloproteinases (MMPs) contributes to tumor angiogenesis and metastasis. NO mediates the enhanced vascular permeability and retention (EPR) effect in solid tumors, and ONOO(-)activates proMMP to MMP in vitro. In this study, we examined the role of ONOO(-)in the EPR effect in solid tumors and normal tissues as related to MMP activation. Authentic ONOO(-), at 50 nmol or higher concentrations, induced the enhanced vascular permeability in normal dorsal skin of mice. ONOO(-)scavengers ebselen and uric acid significantly suppressed the EPR effect in mouse sarcoma 180 (S-180) tumors. Indirect evidence for formation of ONOO(-)in S-180 and mouse colon adenocarcinoma (C-38) tumors included strong immunostaining for nitrotyrosine in the tumor tissue, predominantly surrounding the tumor vessels. MMP inhibitor BE16627B (66.6 mg / kg i.v., given 2 times) or SI-27 (10 mg / kg i.p., given 2 times) significantly suppressed the ONOO(-)-induced EPR effect in S-180 tumors and in normal skin. Soybean trypsin inhibitor (Kunitz type), broad-spectrum proteinase inhibitor ovomacroglobulin, and bradykinin receptor antagonist HOE 140 also significantly suppressed the ONOO(-)-induced EPR effect in normal skin tissues. These data suggest that ONOO(-)may be involved in and promote the EPR effect in tumors, which could be mediated partly through activation of MMPs and a subsequent proteinase cascade to generate potent vasoactive mediators such as bradykinin.

Topics: Adenocarcinoma; Animals; Azoles; Bradykinin; Capillary Permeability; Collagenases; Colonic Neoplasms; Dose-Response Relationship, Drug; Enzyme Precursors; Free Radical Scavengers; Gelatinases; Guinea Pigs; Isoindoles; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Metalloendopeptidases; Mice; Mice, Inbred C57BL; Nitrates; Oligopeptides; Organoselenium Compounds; Protease Inhibitors; Sarcoma 180; Skin; Tyrosine; Uric Acid

Nitric oxide (NO), the production of which is dependent on NO synthase (NOS), has been shown to contribute to various pathogeneses in cancer. The aim of this study was to determine whether inducible NO synthase (iNOS) is overexpressed in human colon carcinoma tissue, and whether NO is produced in tumor tissue.. We investigated iNOS mRNA expression in 24 human colon carcinoma tissue specimens by reverse transcription-polymerase chain reaction (RT-PCR). We then examined the expression of iNOS protein and nitrotyrosine, which indicates NO production in tissue, by immunohistochemistry. The possible immunosuppressive role of NO produced by colon carcinoma cells was analyzed in vitro.. Semiquantitative RT-PCR analysis showed that iNOS mRNA expression in carcinoma tissues is elevated significantly compared to that in noncarcinoma tissue. Immunohistochemistry revealed that iNOS and nitrotyrosine are expressed strongly in carcinoma tissues. In vitro experiments showed that the supernatant from a culture of cytokine-treated colon carcinoma cells, which contained high levels of NO, significantly reduced the phytohemagglutinin (PHA)-stimulated, human lymphocyte proliferative response (60% of the control value).. In human colon carcinoma tissue, iNOS mRNA, protein, and NO products are overexpressed and may contribute to tumor-related immunosuppression.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Colonic Neoplasms; Female; Humans; Immunohistochemistry; Male; Middle Aged; Nitric Oxide; Nitric Oxide Synthase; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sigmoid Neoplasms; Tyrosine

Previous work from our laboratory indicated that the bile salt sodium deoxycholate (NaDOC) induced apoptosis in cultured cells and in normal goblet cells of the colonic mucosa. We also reported that the normal-appearing flat mucosa of patients with colon cancer exhibited apoptosis resistance. Using immunofluorescence in conjunction with confocal microscopy, we now report that high physiological concentrations (0.5 mM) of NaDOC result in the formation of nitrotyrosine residues, a footprint for the formation of reactive nitrogen species, including peroxynitrite, in plasma membrane-associated proteins of HT-29 cells. Because peroxynitrite is formed from the reaction between nitric oxide and superoxide anion, we specifically looked at the role of nitric oxide and superoxide anion in NaDOC-induced apoptosis. Pretreatment of cells with the inhibitor/antioxidants, N-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase, copper (II) 3,5-diisopropyl salicylate hydrate, a superoxide dismutase mimetic compound, and Trolox, a water-soluble analog of alpha-tocopherol, alone or in combination, sensitized cells to apoptosis induced by 0.5 mM NaDOC. These results suggest that nitric oxide may be part of a signaling pathway that is responsible for apoptosis resistance. The results also indicate that nitric oxide does not appear to protect cells against NaDOC-induced apoptosis by scavenging superoxide anion.

Topics: Apoptosis; Bile Acids and Salts; Colonic Neoplasms; Deoxycholic Acid; Enzyme Inhibitors; Fluorescent Antibody Technique; Free Radical Scavengers; Humans; Microscopy, Confocal; NG-Nitroarginine Methyl Ester; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Oxidative Stress; Salicylates; Superoxides; Tumor Cells, Cultured; Tyrosine

An increased expression of nitric oxide synthase (NOS) has been observed in human colon carcinoma cell lines as well as in human gynecological, breast, and central nervous system tumors. This observation suggests a pathobiological role of tumor-associated NO production. Hence, we investigated NOS expression in human colon cancer in respect to tumor staging, NOS-expressing cell type(s), nitrotyrosine formation, inflammation, and vascular endothelial growth factor expression. Ca2+-dependent NOS activity was found in normal colon and in tumors but was significantly decreased in adenomas (P < 0.001) and carcinomas (Dukes' stages A-D: P < 0.002). Ca2+-independent NOS activity, indicating inducible NOS (NOS2), is markedly expressed in approximately 60% of human colon adenomas (P < 0.001 versus normal tissues) and in 20-25% of colon carcinomas (P < 0.01 versus normal tissues). Only low levels were found in the surrounding normal tissue. NOS2 activity decreased with increasing tumor stage (Dukes' A-D) and was lowest in colon metastases to liver and lung. NOS2 was detected in tissue mononuclear cells (TMCs), endothelium, and tumor epithelium. There was a statistically significant correlation between NOS2 enzymatic activity and the level of NOS2 protein detected by immunohistochemistry (P < 0.01). Western blot analysis of tumor extracts with Ca2+-independent NOS activity showed up to three distinct NOS2 protein bands at Mr 125,000-Mr 138,000. The same protein bands were heavily tyrosine-phosphorylated in some tumor tissues. TMCs, but not the tumor epithelium, were immunopositive using a polyclonal anti-nitrotyrosine antibody. However, only a subset of the NOS2-expressing TMCs stained positively for 3-nitrotyrosine, which is a marker for peroxynitrite formation. Furthermore, vascular endothelial growth factor expression was detected in adenomas expressing NOS2. These data are consistent with the hypothesis that excessive NO production by NOS2 may contribute to the pathogenesis of colon cancer progression at the transition of colon adenoma to carcinoma in situ.

Topics: Adenoma; Blotting, Western; Carcinoma; Colon; Colonic Neoplasms; Disease Progression; DNA Primers; DNA, Neoplasm; Endothelial Growth Factors; Endothelium, Vascular; Humans; Immunohistochemistry; Lymphokines; Neoplasm Proteins; Neovascularization, Pathologic; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Phosphorylation; Polymerase Chain Reaction; Tyrosine; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors