Dual role of NO donors in the reversal of tumor cell resistance and EMT: Downregulation of the NF-κB/Snail/YY1/RKIP circuitry.

Several studies have implicated the role of Nitric Oxide (NO) in the regulation of tumor cell behavior and have shown that NO either promotes or inhibits tumorigenesis. These conflicting findings have been resolved, in part, by the levels of NO used such that low levels promote tumor growth and high levels inhibit tumor growth. Our studies have focused on the use of high levels of NO provided primarily by the NO donor, DETANONOate. We have shown that treatment of resistant tumor cells with DETANONOate sensitizes them to apoptosis by both chemotherapeutic drugs and cytotoxic immunotherapeutic ligands. The underlying mechanisms by which NO sensitizes tumor cells to apoptosis were shown to be regulated, in part, by NO-mediated inhibition of the NF-κB survival/anti-apoptotic pathways and downstream of NF-κB by inhibition of the transcription factor Yin Yang 1 (YY1). In addition to NO-induced sensitization to apoptosis, we have also shown that NO induced the expression of the metastasis-suppressor/immunosurveillance cancer gene product, Raf-1 kinase inhibitor protein (RKIP). Overexpression of RKIP mimics NO in tumor cells-induced sensitization to apoptosis. The induction of RKIP by NO was the result of the inhibition of the RKIP repressor, Snail, downstream of NF-κB. These findings established the presence of a dysregulated NF-κB/Snail/YY1/ RKIP circuitry in resistance and that treatment with NO modifies this loop in tumor cells in favor of the inhibition of tumor cell survival and the response to cytotoxic drugs. Noteworthy, the NF-κB/Snail/YY1/RKIP loop consists of gene products that regulate the epithelial to mesenchymal transition (EMT) and, thus, tumor metastasis. Hence, we have found that treatment of metastatic cancer cell lines with DETANONOate inhibited the EMT phenotype, through both the inhibition of the metastasis-inducers, NF-κB and Snail and the induction of the metastasis-suppressor, RKIP. Altogether, the above findings establish, for the first time, the dual role of high levels of NO in the sensitization of tumor cells to apoptotic stimuli as well as inhibition of EMT. Hence, NO donors may be considered as novel potential therapeutic agents with dual roles in the treatment of patients with refractory cancer and in the prevention of the initiation of the metastatic cascade via EMT.

Pharmacokinetics of 1-nitrosomelatonin and detection by EPR using iron dithiocarbamate complex in mice.

The N-nitroso-derivative of melatonin, NOM (1-nitrosomelatonin), which has been demonstrated to be a NO* [oxidonitrogen*] donor in buffered solutions, is a new potential drug particularly in neurological diseases. The advantage of NOM, a very lipophilic drug, is its ability to release both melatonin and NO*, an easily diffusible free radical. In order to evaluate the distribution and the pharmacokinetics of NOM, [O-methyl-3H]NOM was administered to and followed in mice. A complementary method for monitoring NOM, EPR, was performed in vitro and ex vivo with (MGD)2-Fe2+ (iron-N-methyl-D-glucamine dithiocarbamate) complex as a spin trap. The behaviour of NOM was compared with that of GSNO (S-nitrosoglutathione), a hydrophilic NO* donor. In the first minutes following [O-methyl-3H]NOM intraperitoneal injection, the radioactivity was found in organs (6% in the liver, 1% in the kidney and 0.6% in the brain), but not in the blood. In both liver and brain, the radioactivity content decreased over time with similar kinetics reflecting the diffusion and metabolism of NOM and of its metabolites. Based on the characterization and the quantification of the EPR signal in vitro with NOM or GSNO using (MGD)2-Fe2+ complex in phosphate-buffered solutions, the detection of these nitroso compounds was realized ex vivo in mouse tissue extracts. (MGD)2-Fe2+-NO was observed in the brain of NOM-treated mice in the first 10 min following injection, revealing that NOM was able to cross the blood-brain barrier, while GSNO was not.

Blocking the endogenous formation of N-nitroso compounds and related carcinogens.

Humans are exposed to a wide range of nitrogen-containing compounds and nitrosating agents, such as nitrite, nitrate and nitrogen oxides (NOx), that can react in vivo to form potentially carcinogenic N-nitroso compounds (NOCs), as well as several carcinogenic C-nitro(so) or reactive diazo compounds. Nitrosating agents can also be synthesized endogenously by bacteria and activated macrophages via the nitric oxide (NO) synthase pathway. Endogenous nitrosation can thus occur at many locations in the body, including sites of chronic infection or inflammation. Ascorbic acid, alpha-tocopherol, phenolic compounds, and fruit, vegetable and plant extracts inhibit NOC formation by destroying nitrosating agents. Fresh fruits and vegetables (sources of nitrosation inhibitors) exert a protective effect against various epithelial cancers. In addition to giving rise to nitrosating agents, overproduction of NO in chronic inflammatory conditions leads to the generation of peroxynitrite (a source of oxidative DNA damage and lipid peroxidation) and aldehydes and epoxides derived from lipid peroxidation that yield miscoding exocyclic DNA adducts. Inhibition of the inducible NO synthase and strengthening of the cellular defence system against oxidative stress should block NO-associated DNA damage. This chapter summarizes mechanistic, experimental and human studies that address the inhibition of endogenous formation of NOCs and related carcinogens as a method of chemoprevention.

Dietary garlic suppresses DNA adducts caused by N-nitroso compounds.

The present studies examined the impact of a processed garlic powder on the in vivo occurrence of DNA adducts caused by N-nitroso compounds (NOC) in rats. Addition of 2% garlic powder to diets containing aminopyrine and sodium nitrite (each at 600 mg/kg) reduced the occurrence of both 7-N-methyldeoxyguanosine (7-N-mG) and 6-O-methyldeoxyguanosine (6-O-mG) adducts to rat liver DNA by approximately 55%; and over 80% when 4% garlic was provided. Dietary supplementation with garlic powder (2 and 4%) also reduced the occurrence of 7-N-mG and 6-O-mG adducts by approximately 40 and 60% respectively, in rats intubated with N-nitrosodimethylamine (150 mg/kg body wt). The quantity of 7-N-mG and 6-O-mG adducts in mammary tissue of rats given intravenous N-methyl-N-nitrosourea (50 mg/kg body wt) was reduced over 50% in rats fed 2% garlic compared to controls. The depression in the occurrence of these adducts was approximately 70% when dietary garlic was increased to 4%. These experiments suggest the reduction in DNA adducts caused by processed garlic powder likely reflects a depression in the formation of NOC from precursors and changes in the bioactivation and/or denitrosation of NOC.

In vivo nitrosoproline formation and other risk factors in Costa Rican children from high- and low-risk areas for gastric cancer.

The hypothesis that intragastric synthesis of N-nitroso compounds (NOC) in early life could play a role in gastric carcinogenesis was tested by applying the N-nitrosoproline (NPRO) test to about 50 children living in high- and low-risk areas for stomach cancer in Costa Rica. The median values of excretion of NPRO and the sum of three nitrosamino acids (micrograms/12 h urine) were 10-20% of those in adults from other geographical high-risk areas for stomach cancer. The urinary NPRO level after proline intake was higher in children from the high-risk area (P < 0.04) and markedly reduced after ingestion of ascorbic acid together with proline (P < 0.05). NPRO levels on the day of proline intake were highly correlated with levels of nitrate excretion (P < 0.001). Mean levels of total NOC in an aqueous (pH 2) extract of cooked beans from the high- and low-risk areas were similar. Acid-catalyzed nitrosation of the extract increased the total NOC concentration up to 1000-fold, but there was no difference between samples from the two areas. About 10% of bean extracts from both areas showed weak direct-acting genotoxicity in Escherichia coli; after acid-catalyzed nitrosation, all samples were genotoxic at similar levels. The diet of children in the low-risk area satisfied recommended levels of intake of energy and most nutrients except riboflavin and retinol equivalents. Diets from the high-risk area were deficient in energy intake and all nutrients except protein and vitamin C.(ABSTRACT TRUNCATED AT 250 WORDS)

Endogenous nitrosation in the oral cavity of chewers while chewing betel quid with or without tobacco.

In order to evaluate endogenous nitrosation in the oral cavity of chewers of betel quid with tobacco (BQT) or without tobacco (BQ), saliva samples were collected from healthy male volunteers after chewing sequentially (i) unmodified BQT or BQ, (ii) BQT or BQ to which proline has been added, and (iii) BQT or BQ to which proline and ascorbic acid had been added. Samples were collected over 20 min and analysed for N-nitrosoproline (NPRO), tobacco-specific nitrosamines (TSNA) and areca nut-specific nitrosamines using gas chromatography-thermal energy analysis, arecoline and nicotine using gas chromatography-nitrogen phosphorus-specific detector, and for nitrite and thiocyanate. When results were expressed as a ratio of NPRO (ng/ml) to nicotine (micrograms/ml), all BQT chewers had increased NPRO contents after chewing BQT with proline. For BQ chewers, when the results were expressed as a ratio of NPRO (ng/ml) to arecoline (micrograms/ml), a similar increase in NPRO content was observed. However, the presence of ascorbic acid inhibited the increased nitrosation in only four out of ten BQT chewers and in five out of ten BQ chewers; in the rest of the samples, its presence enhanced the levels of NPRO. N'-Nitrosoanatabine (NAT) and N-nitrosoguvacoline (NGCO) levels decreased significantly in saliva of chewers of BQT in the presence of ascorbic acid, suggesting inhibition of their formation. In-vitro nitrosation of BQT/BQ with proline and proline plus ascorbic acid showed a similar pattern of nitrosation at salivary pH. The study confirmed previous results that certain nitrosamines are formed during the chewing of BQT/BQ.