Neoplastic transformation and induction of H+,K+-adenosine triphosphatase by N-methyl-N′-nitro-N-nitrosoguanidine in the gastric epithelial RGM-1 cell line

N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) induces gastric cancer in animal models. We established an MNNG-induced mutant of the rat murine RGM-1 gastric epithelial cell line, which we named RGK-1, that could be used as an in vitro model of gastric cancer. This cell line showed signs of neoplasia and transformation, in that it lost contact inhibition and formed tumors in nude mice. The mutant cells also expressed parietal cell-specific H⁺,K⁺-adenosine triphosphatase (H⁺,K⁺-ATPase), which parent RGM-1 did not. The results suggested that parent RGM-1 cells were gastric progenitor cells. This mutant RGK-1 cell line will contribute to future investigation on gastric carcinogenesis and to the development of other pathophysiologic fields.     

Helicobacter pylori infection can modulate the susceptibility of gastric mucosa cells to MNNG

The pathogenesis of stomach cells can be associated with their susceptibility to exogenous dietary irritants, like nitrosamines such as dimethylnitrosamines (DMNA), and to the effects of non-dietary factors, including Helicobacter pylori infection. We used N-methyl-N’-nitro N-nitrosoguanidyne (MNNG) as a surrogate agent that induces a spectrum of DNA damage similar to DMNA. Using the alkaline comet assay, we showed that antioxidants — vitamins C and E, quercetin, and melatonin — reduced the genotoxic effect of MNNG in H. pylori-infected and non-infected human gastric mucosa cells (GMCs). To compare the sensitivity of the stomach and the blood, the experiment was also carried out in peripheral blood. We observed a higher level of DNA damage induced by MNNG in H. pylori-infected than in noninfected GMCs. We did not note any difference in the efficacy of the repair of the damage in either type of GMC. H. pylori infection may play an important role in the pathogenesis of GMCs, as it can modulate their susceptibility to dietary mutagens/carcinogens, thus contributing to gastric cancer.     

Evaluation of Multi-Target and Single-Target Liposomal Drugs for the Treatment of Gastric Cancer

We studied the effects of multi- and single-target liposomal drugs on human gastric cancer cell AGS both in vitro and in vivo. The cytotoxic effect of dihydrotanshinone I was significantly enhanced by treatment with octreotide-polyethylene glycol(PEG)-liposome, Arg-Gly-Asp(RGD)-PEG-liposome, and RGD/octreotide-PEG-liposome encapsulated with 0.5 μg/ml of dihydrotanshinone I to AGS cell for 24 h, compared to control. Furthermore, the AGS cell survival rate for multi-target versus single target liposomal drugs was significantly suppressed. Microsocpic examination revealed that significant cell death occurred in the multi- and single-target liposomal encapsulated drug groups. Significant suppression of tumor growth in AGS cell xenograft nude mice given octreotide-PEG-liposome, RGD/octreotide-PEG-liposome encapsulated drug, versus those given a free drug was noted after 13 d of experimentation with the multi-targeted liposome: up to 60.75% and 41.2% reduction of tumor volume as compared to dimethylsulfoxide (DMSO) control and the free drug groups respectively. The treated animals showed no gross signs of toxicity. The results have potential clinical application.     

Linker-based GnRH-PE chimeric proteins inhibit cancer growth in nude mice

Since the number of cancer-related deaths has not decreased in recent years, major efforts are being made to find new drugs for cancer treatment. In this report we introduce the gonadotropin releasing hormone-Pseudomonas exotoxin (GnRH-PE) based chimeric proteins L-GnRH-PE66 and L-GnRH-PE40. These proteins are composed of a GnRH moiety attached to modified forms ofPseudomonas exotoxin via a polylinker (gly₄ser)₂. The chimeric proteins L-GnRH-PE66 and L-GnRH-PE40 have the ability to target and kill adenocarcinoma cell linesin vitro, whereas non-adenocarcinoma cell lines are not affected. We demonstrate that L-GnRH-PE66 and L-GnRH-PE40 efficiently inhibit cancer growth. Nude mice were injected subcutaneously with the SW-48 adenocarcinoma cell line to produce xenograft tumours. When the tumours were established and visible, the animals were injected with chimeric proteins for 10 days. At the end of this period, a reduction of up to 3-fold in tumor size was obtained in the treated mice, as compared with the control group, which received equivalent amounts of GnRH; the difference was even greater 13 days after termination of treatment. Thus, the chimeric proteins L-GnRH-PE66 and L-GnRH-PE40 are promising candidates for treatment of a variety of adenocarcinomas and their use in humans should be considered.     

Alteration in N-acetylglucosaminyltransferase activities and glycan structure in tissue and bile glycoproteins from extrahepatic bile duct carcinoma

The activities of three N-acetylglucosaminyltransferases (GnT)-III, IV and V, as well as the structural alterations of N-glycans on the glycoproteins in cancer tissues and bile specimens from 28 cases of extrahepatic bile duct carcinoma (EBDC) were compared with those from 18 cases of benign biliary duct diseases (BBDD). GnT activities were determined with fluorescence-labeled substrate using a HPLC method. It was found that GnT-III and GnT-V activities in EBDC were increased to 3.14 and 15.96 times respectively of the mean BBDD values, but GnT-IV remained unchanged. The activity of GnT-V was correlated with the grade of differentiation and TMN stage of EBDC. The up-regulation of GnT-III resulted in the increased bisecting-GlcNAc on the N-glycans of glycoproteins in cancer tissues and a 201 kDa bile glycoprotein when analyzed with HRP-labeled E₄-PHA. The increased GnT-V activity led to the elevation of the β1,6GlcNAc branch (or antennary number) on the N-glycans in cancer tissue glycoproteins and 201, 163, 122 kDa proteins in the bile as probed with HRP-labeled DSA. These findings suggest that the alteration in GnT activities may be involved in the malignant transformation and development of EBDC, resulting in the aberrant glycosylation of some tissue and bile proteins. The latter was expected to be used in the clinical diagnosis and prognosis evaluation in EBDC patients. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

Development and characterization of a potent producer of penicillin G amidase by mutagenization

Summary The penicillin G amidase (PGA) activity of a parent strain of E. coli (PCSIR-102) was enhanced by chemical mutagenization with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). After screening and optimization, a penicillinase deficient mutant (MNNG-37) was isolated and found effective for the production of penicillin G amidase as compared to the parent strain of E. coli (PCSIR-102). Penicillin G amidase activity of MNNG-37 appeared during an early stage of growth, whereas PCSIR-102 did not exhibit PGA activity due to the presence of penicillinase enzyme which inhibits the activity of enzyme PGA. However, MNNG-37 gave a three-fold increase in enzyme activity (231 IU mg⁻¹) as compared to PCSIR-102 (77 IU mg⁻¹) in medium containing 0.15 and 0.1% concentrations of phenylacetic acid, respectively which was added after 6 h of cultivation. The difference in K _m values of the enzyme produced by parent strain PCSIR-102 (0.26 mM) and mutant strain MNNG-37 (0.20 mM) is significant (1.3-fold increase in K _m value) which may show the superiority of the latter in terms of better enzyme properties.     

Morphological,growth, and chromosomal changes in bovine pancreatic duct epithelial cells exposed toN-methyl-N′-nitro-N-nitrosoguanidine

Summary Epithelial cells cultured from bovine pancreatic ducts were given a single treatment ofN-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Multinucleated cells and giant cells were observed more frequently in carcinogen-treated cultures than in controls. The MNNG-treated cultures also contained a sizeable population of small, dense cells that were not observed in control cultures. At the concentration of 1.0 μg/ml, MNNG caused an initial depression in the growth rate of the cells followed by growth stimulation for several weeks. The MNNG produced chromosomal damage in the cells as indicated by the observation that a substantial proportion of carcinogen-treated cells were heteroploid and contained a high frequency of metacentric and submetacentric chromosomes and a dicentric marker chromosome. The MNNG treated and control cultures did not acquire the ability to grow in soft agar or to produce tumors after transplantation into athymic, nude mice. This work was supported in part by Public Health Service Contract and Interagency Agreement Y01-CP60204 from the Division of Cancer Cause and Prevention, National Cancer Institute.     

Induction of kinetochore positive and negative micronuclei in V79 cells by N-methyl-N′-nitro-N-nitrosoguanidine: the protective effect of the pyridoindole antioxidant stobadine

The induction of micronuclei by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and their reduction by the cardioprotective synthetic antioxidant, stobadine were studied in hamster V79 cells cultured in vitro. The micronuclei derived from acentric fragments or from whole chromosomes were evaluated with the help of an immunofluorescent staining using antikinetochore antibodies from the serum of scleroderma (CREST syndrome) patients. Our results showed that MNNG (0.5 μg/ml) induced mainly kinetochore-negative micronuclei. At 6, 24 and 48 h after MNNG treatment, we measured a 2.7-, 4.3- and 7.0-fold increase, respectively, of kinetochore-negative micronuclei over the controls. The increase of kinetochore-positive micronuclei was rather low and represented at 6, 24 and 48 h, respectively 0.9-, 1.8- and 2.6-fold increases over the controls. Stobadine decreased the level of kinetochore-negative micronuclei at 6, 24 and 48 h to approximately one-half; the frequency of kinetochore-positive micronuclei was reduced only at 6 h. We suppose that the antioxidant stobadine reduces the induction of micronuclei by MNNG by scavenging of MNNG-induced highly reactive OH radicals which cause chromosomal damage.     

Analysis of urinary N-acetyl-S-(N-methylcarbamoyl)cysteine, the mercapturic acid derived from N,N-dimethylformamide

Human biotransformation of the industrial solvent N,N-dimethylformamide gives raise to N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC) which has the longest half-life (about 23 h) among urinary metabolites of N,N-dimethylformamide. It could be used for monitoring industrial exposure over several workdays, by measuring it in urine samples collected at the end of the working week. This is consistent with the suggestions of the American Conference of Governmental Industrial Hygienists, which established a limit of 40 mg/l for the year 2000. An easy, cheap and user-friendly method has been developed for determination of urinary AMCC. Unlike currently available methods, it requires neither a time-consuming preparation phase nor gas chromatographic analysis with a nitrogen-phosphorus or mass detector. The method uses high-performance liquid chromatography (HPLC), with an UV detector at 436 nm. A 10-μl volume of urine is added to a carbonate–hydrogen carbonate buffer and mixed with a dabsyl chloride solution in acetonitrile. The reaction between AMCC and the reagent is performed at 70°C for 10 min. The ‘dabsylated’ product is stable for at least 12 h. After brief centrifugation, the solution is ready for HPLC analysis using a C₁₈ column (250×4.6 mm, 5 μm). The method is sensitive (detection limit 1.8 mg/l) and specific. It identified urinary AMCC in urine of 40 subjects not exposed to N,N-dimethylformamide with a median concentration of 3.9 mg/l. In urine samples from 20 workers exposed to N,N-dimethylformamide (5–40.8 mg/m³), AMCC concentrations ranged from 16 to 170 mg/l. Industrial toxicology laboratories with limited instrumentation will be able to use it in the biological monitoring of workers exposed to N,N-dimethylformamide.     

Chronic CagA-positive Helicobacter pylori infection with MNNG stimulation synergistically induces mesenchymal and cancer stem cell-like properties in gastric mucosal epithelial cells

A CagA-positive Helicobacter pylori (H. pylori) infection can cause malignant transformation of human gastric mucosal epithelial cells, and N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) is a chemical carcinogen that induces gastric carcinogenesis. Whether this environmental chemocarcinogen may synergistically enhance the risk of H. pylori-infected gastric cancer remains unclear. In this study, we adopted a chronic CagA-positive H. pylori infection with or without MNNG coinduction to establish a cellular model in GES-1 cells and an animal model in C57BL/6J mice. The proliferation, cell phenotype, apoptosis, epithelial-mesenchymal transition (EMT), stemness and tumorigenicity of gastric mucosal epithelial cells were analyzed in vitro and in vivo. The results showed that chronic H. pylori-infected GES-1 cells displayed inhibited apoptosis, abnormal proliferation, enhanced invasion, and migration, increased EMT/mesenchymal phenotype, colony formation and stem cell-like properties, and enhanced tumorsphere-formatting efficiency as well as CD44 expression, a known gastric cancer stem cell (CSC) marker. MNNG synergistically promoted the above actions of chronic H. pylori infection. Further studies in chronic H. pylori-infected C57BL/6J mice models showed that an increased incidence of premalignant lesions in the gastric mucosa tissue of the H. pylori-infected mice had occurred, the mouse gastric mucosa cells exhibited similar mesenchymal and CSC-like properties in the above GES-1 cells, and precancerous lesions and EMT/CSC-like phenotypes were reinforced by the synergistic action of MNNG stimulation. H. pylori infection and/or MNNG induction were capable of causing enhanced expression and activation of Wnt2 and β-catenin, indicating that the Wnt/β-catenin pathway is involved in the actions of H. pylori and MNNG. Taken together, these findings suggest that chronic CagA-positive H. pylori infection with MNNG stimulation synergistically induces mesenchymal and CSC-like properties of gastric mucosal epithelial cells.     

TIPE1 suppresses invasion and migration through down‐regulating Wnt/β‐catenin pathway in gastric cancer

Epithelial–mesenchymal transition (EMT) plays an important role in the invasiveness and metastasis of gastric cancer. Therefore, identifying key molecules involved in EMT will provide new therapeutic strategy for treating patients with gastric cancer. TIPE1 is a newly identified member of the TIPE (TNFAIP8) family, and its contributions to progression and metastasis have not been evaluated. In this study, we found that the levels of TIPE1 were significantly reduced and inversely correlated with differentiation status and distant metastasis in primary gastric cancer tissues. We further observed overexpression of TIPE1 in aggressive gastric cancer cell lines decreased their metastatic properties both in vitro and in vivo as demonstrated by markedly inhibiting EMT and metastasis of gastric cancer cells in nude mice. Consistently, gene silencing of TIPE1 in well‐differentiated gastric cancer cell line (AGS) inhibited these processes. Mechanistically, we found that TIPE1‐medicated Wnt/β‐catenin signalling was one of the critical signal transduction pathways that link TIPE1 to EMT inhibition. Importantly, TIPE1 dramatically restrained the expression and activities of MMP2 and MMP9 which are demonstrated to promote tumour progression and are implicated in EMT. Collectively, these findings provide new evidence for a better understanding of the biological activities of TIPE1 in progression and metastasis of gastric cancer and suggest that TIPE1 may be an innovative diagnostic and therapeutic target of gastric cancer.     

Concise synthesis of 4-methylumbelliferyl-penta-N-acetylchitopentaoside and its inhibition effect on chitinase

The synthesis of 4-methylumbelliferyl (UMB)-penta-N-acetylchitopentaoside 4 and its inhibition effect on chitinase are described. The fluorophore-assisted carbohydrate electrophoresis (FACE) analysis showed that the partially N-acetylated chitooligosaccharide (COS) mixture mainly contained glucosamine (GlcN) and oligomers [(GlcN)_n, n = 2–7]. The peracetylated COSs [(GlcNAc)_n, n = 1–7] were synthesized by treating the partially N-acetylated COS mixture with Ac₂O–NaOAc. The peracetylated chitopentaoside 1 was obtained by isolation of peracetylated COS mixture. The peracetylated UMB chitopentaoside 3 was synthesized by treating compound 1 with 4-methylumbelliferone and a Lewis acid (SnCl₄) catalyst. NaOMe in dry methanol was used for deacetylation of the blocked derivative, to give the target compound 4 in an overall yield of 32%. In binding chitinase assay, it indicates that compound 4 is much more stable than the corresponding penta-N-acetylchitopentaose 2.     

Decomposition of N-nitrosamines, and concomitant release of nitric oxide by Fenton reagent under physiological conditions

N-Nitrosodimethylamine (NDMA) in phosphate buffer was rapidly decomposed by Fenton reagent composed of H₂O₂, and Fe(II) ion. Electron spin resonance (ESR) studies using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) showed that characteristic four line 1:2:2:1 ESR signals due to the DMPO-OH adduct formed on treatment of DMPO with Fenton reagent disappeared in the presence of NDMA, and N-nitrosodiethylamine (NDEA), suggesting the interaction of the N-nitrosamines with Fenton reagent. Treatment of the N-nitrosamines with Fenton reagent generated nitric oxide (NO) as estimated by ESR technique using cysteine–Fe(II), and N-methyl- -glucaminedithiocarbamate (MGD)–Fe(II) complexes. Characteristic 3, and single line signals due to 2 cysteine–Fe(II)–NO, and 2 cysteine–Fe(II)–2 NO complexes, respectively, and three line signals due to MGD–Fe(II)–NO were observed. Considerable amount of NO were liberated as determined by NO₂⁻, the final oxidation product of NO formed by reaction with dissolved oxygen in the aqueous medium. Spontaneous release of a small amount of NO from the N-nitrosamines was observed only on incubation in neutral buffers. Above results indicate that the N-nitrosamines were decomposed accompanying concomitant release of NO on contact with reactive oxygen species.     

Expression of apurinic/apyrimidinic endonuclease-1 (APE-1) in H. pylori-associated gastritis, gastric adenoma, and gastric cancer

Background and Aim: Apurinic/apyrimidinic endonuclease‐1 (APE‐1) is a key enzyme in DNA base excision repair (BER), linked to cancer chemosensitivity. However, little is known about the localization of APE‐1 in Helicobacter pylori‐infected gastric mucosa or its role in the development of gastric cancer. To investigate the role of APE‐1 in the development of gastric cancer, we examined APE‐1 expression and localization in cultured cells and gastric biopsies from patients with H. pylori‐infected gastritis or gastric adenoma, and from surgically resected gastric cancer. Methods: APE‐1 mRNA and protein expression were determined in H. pylori (CagA+) water‐extract protein (HPWEP)‐stimulated MKN‐28 cells, gastric adenocarcinoma cell‐line (AGS) cells, and human peripheral macrophages by real‐time polymerase chain reaction and Western blot analysis. APE‐1 expression and 8‐OHdG as a measure of oxidative DNA damage were evaluated by immunostaining. Localization of APE‐1 and IκBα phosphorylation in gastric adenoma and gastric cancer tissues were evaluated by single‐ and double‐label immunohistochemistry. Results: In studies in vitro, HPWEP‐stimulation significantly increased APE‐1 mRNA expression levels in both MKN‐28 cells and human peripheral macrophages. Hypo/reoxygenation treatment significantly increased APE‐1 protein expression in HPWEP‐stimulated MKN‐28 cells. HPWEP stimulation significantly increased both APE‐1 expression and IκBα phosphorylation levels in MKN‐28 and AGS cells. In human tissues, APE‐1 expression in H. pylori‐infected gastritis without goblet cell metaplasia was significantly increased as compared to that in tissues from uninfected subjects. Eradication therapy significantly reduced both APE‐1 and 8‐OHdG expression levels in the gastric mucosa. APE‐1 expression was mainly localized in epithelial cells within gastric adenoma and in mesenchymal cells of gastric cancer tissues. APE‐1 expression in gastric cancer tissues was significantly reduced compared to that in H. pylori‐infected gastric adenoma, while 8‐OHdG index and IκBα phosphorylation levels did not differ between these two neoplastic tissue types. Co‐localization of APE‐1 and IκBα phosphorylation was observed not in gastric cancer cells but in gastric adenoma cells. Conclusion: H. pylori infection is associated with increased APE‐1 expression in human cell lines and in gastric tissues from subjects with gastritis and gastric adenomas. The observed distinct expression patterns of APE‐1 and 8‐OHdG in gastric adenoma and gastric cancer tissues may provide insight into the progression of these conditions and warrants further investigation.     

Systematic analysis of acid, neutral and basic drugs in horse plasma by combination of solid-phase extraction, non-aqueous partitioning and gas chromatography–mass spectrometry

A sample preparation method for mass chromatographic detection of doping drugs from horse plasma is described. Bond Elut Certify (1 g/6 ml) is used for the extraction of 4 ml of horse plasma. Fractionation is performed with 6 ml of CHCl₃–Me₂CO (8:2) and 5 ml of 1% TEA–MeOH according to its property. Simple and effective clean-up based on non-aqueous partitioning is adopted to remove co-eluted contaminants in both acid and basic fractions. Two kinds of 1-(N,N-diisopropylamino)-n-alkanes are co-injected with the sample into the GC–MS system for the calculation of the retention index. Total recoveries of 107 drugs are examined. Some data of post administration plasma are presented. This procedure achieves sufficient recoveries and clean extracts for GC–MS analysis. The method is able to detect ng/ml drug levels in horse plasma.     

High-performance liquid chromatographic determination of [C]1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide in mouse plasma and tissue and in human plasma

The high-performance liquid chromatographic determination of 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide ([¹¹C]PK 11195) is described. The method was successfully applied for plasma and tissue analysis after i.v. injection of [¹¹C]PK 11195 in mice and for plasma analysis after administration of [¹¹C]PK 11195 to humans. Separation is effected on a RP-C₁₈ column, using a mixture of acetonitrile–water–triethylamine (65:35:0.5, v/v). Quantitative measurements of radioactivity are performed on a one-channel γ-ray spectrometer equipped with a 2×2 in. NaI(Tl) detector. For humans rapid metabolisation of [¹¹C]PK 11195 was observed. At 5, 20 and 35 min post injection 5%, 22% and 32%, respectively, of the plasma activity consisted of at least two more polar metabolites. Despite the extensive metabolisation rate in mice (up to 42% at 10 min post injection of [¹¹C]PK 11195), no ¹¹C-labelled metabolites could be detected in the extracts of brain and heart.     

Low incidence of N-glycolylneuraminic acid in birds and reptiles and its absence in the platypus

The sialic acids of the platypus, birds, and reptiles were investigated with regard to the occurrence of N-glycolylneuraminic (Neu5Gc) acid. They were released from tissues, eggs, or salivary mucin samples by acid hydrolysis, and purified and analyzed by thin-layer chromatography, high-performance liquid chromatography, and mass spectrometry. In muscle and liver of the platypus only N-acetylneuraminic (Neu5Ac) acid was found. The nine bird species studied also did not express N-glycolylneuraminic acid with the exception of an egg, but not tissues, from the budgerigar and traces in poultry. Among nine reptiles, including one turtle, N-glycolylneuraminic acid was only found in the egg and an adult basilisk, but not in a freshly hatched animal. BLAST analysis of the genomes of the platypus, the chicken, and zebra finch against the CMP-N-acetylneuraminic acid hydroxylase did not reveal the existence of a similar protein structure. Apparently monotremes (platypus) and sauropsids (birds and reptiles) cannot synthesize Neu5Gc. The few animals where Neu5Gc was found, especially in eggs, may have acquired this from the diet or by an alternative pathway. Since Neu5Gc is antigenic to man, the observation that this monosaccharide does not or at least only rarely occur in birds and reptiles, may be of nutritional and clinical significance.     

Analysis of N-methylhistamine by gas chromatography–mass spectrometry

A gas chromatography–electron capture mass spectrometry assay has been developed for the histamine H₃ receptor agonist, N^α-methylhistamine (N^α-MH). The assay is linear from 50 pg–10 ng, with a limit of detection of 50 pg/ml for gastric juice and plasma, and 50 pg/sample for bacteria (10⁷–10⁸ CFU) and gastric tissue (5–10 mg wet weight). The limits of quantification are 100 pg/ml for gastric juice (%RSD=1.4) and plasma (%RSD=9.4), and 100 pg/sample for bacteria (%RSD=3.9) and tissue (%RSD=5.8). N^α-MH was not present in human plasma, but low levels (1.4 ng/ml and 0.4 ng/ml) were detected in two samples of human gastric juice obtained from patients infected with Helicobacter pylori.