Suppression of acute experimental colitis by a highly selective inducible nitric-oxide synthase inhibitor, N-[3-(aminomethyl)benzyl]acetamidine.

High concentrations of nitric oxide (NO) produced by the inducible nitric-oxide synthase (iNOS) are associated with ulcerative inflammation and disease activity in colitis. Therefore, inhibition of iNOS serves as a novel experimental approach to treat gut inflammation. The aim of the present study was to investigate the effects of a novel highly selective iNOS inhibitor, N-[3-(aminomethyl)benzyl]acetamidine (1400W), as compared with a nonselective NOS inhibitor, N(G)-nitro-L-arginine-methyl-ester (L-NAME), in 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced acute colitis in the rat. Increased expression of iNOS protein and mRNA was found in acute TNBS-induced colitis along with neutrophil infiltration, inflammatory edema, and tissue damage. In a 24-h model of acute colitis, subcutaneous injections of 1400W (5 or 10 mg/kg t.i.d.) produced a 56 and 95% reduction in inflammatory edema formation, a 68 and 63% reduction in neutrophil infiltration (measured as myeloperoxidase activity), and a 19 and 26% decrease in the size of mucosal lesions as compared with vehicle treatment. Administration of L-NAME (35 mg/kg) failed to produce any significant beneficial effects as compared with vehicle treatment in this experimental model of acute colitis. Treatment with 1400W, a highly selective inhibitor of iNOS, reduced formation of edema, neutrophil infiltration, and macroscopic inflammatory damage in experimentally induced acute colitis in the rat. In contrast, nonselective nitric-oxide synthase inhibition with L-NAME provided no benefit. These results support the idea that selective iNOS inhibitors have a promise in the treatment of colitis.

Bi-directional effects of the elevation of intracellular calcium on the expression of inducible nitric oxide synthase in J774 macrophages exposed to low and to high concentrations of endotoxin.

Nitric oxide produced through the action of inducible nitric oxide synthase (iNOS) is an important mediator in immune responses of the host. Various extracellular factors, including inflammatory stimuli, affect intracellular free Ca2+ levels ([Ca2+](i)), modulating cellular signalling and gene expression. In the present study we investigated the effects of increased ([Ca2+](i)) on NO production through the iNOS pathway in J774 macrophages. Thapsigargin (TG), a Ca2+-ATPase inhibitor, and the Ca2+ ionophore A23187 were used as tools to induce an increase in ([Ca2+](i)) in the cytosol. This increase was confirmed by the fura 2 method. The production of NO was measured as accumulated nitrite in the cell culture medium; iNOS protein and iNOS mRNA were detected by Western blotting and reverse-transcriptase-mediated PCR respectively. The activation of nuclear factor kappaB (NF-kappaB) was investigated by electrophoretic mobility-shift assay. TG (100 nM) induced a marked synthesis of iNOS mRNA, iNOS protein and NO in cells primed with a low concentration of endotoxin [lipopolysaccharide (LPS) 1 ng/ml], which on its own induced barely detectable NO synthesis. Stimulation by a high concentration of LPS (100 ng/ml) induced a marked expression of iNOS and NO production. Under these conditions, treatment with TG hindered the synthesis of iNOS protein and NO production by accelerating the degradation of iNOS mRNA. Treatment with TG (100 nM) did not affect the NF-kappaB activity induced by low (1 ng/ml) or high (100 ng/ml) concentrations of LPS. Viability of the cells was confirmed by the 2,3-bis[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5-carboxyaniline ("XTT") method; apoptosis was ruled out by propidium iodide staining and flow cytometry. A23187 (1 microM) also transiently increased ([Ca2+](i)) and had opposite effects on NO production depending on the LPS concentration. Our results show that increased ([Ca2+](i)) induced the stimulation or suppression of NO production through iNOS in macrophages depending on the state of cell activation. These findings suggest that the receptor-mediated increase in ([Ca2+](i)) might be an important factor in the control of the balance between the up-regulation and down-regulation of inflammatory genes, including that encoding iNOS, depending on the phase of the inflammatory response.

Inhibition of extracellular signal-regulated kinase suppresses endotoxin-induced nitric oxide synthesis in mouse macrophages and in human colon epithelial cells.

Macrophages produce large amounts of nitric oxide (NO) in response to proinflammatory cytokines and lipopolysaccharide (LPS) by expressing inducible isoform of NO synthase (iNOS). We examined the role of extracellular signal-regulated kinase p42/44(MAPK) (Erk1/2) in signal transduction pathways leading to induction of NO synthesis in response to LPS in J774 mouse macrophages and T-84 human colon epithelial cells. LPS activated Erk1/2 and induced iNOS and subsequent NO production. Erk1/2 activation was inhibited by PD 98059, a specific inhibitor of mitogen-activated protein kinase kinase (Mek) that is an upstream activator of Erk1/2. At corresponding concentrations PD 98059 reduced LPS-induced NO formation by 40 to 50% by inhibiting iNOS expression in J774 and T-84 cells. Inhibition of iNOS expression was not mediated by nuclear factor-kappaB because PD 98059 had no effect on nuclear factor-kappaB activity in J774 macrophages. In addition, PD 98059 reduced LPS-induced L-arginine transport into the cells as measured in J774 macrophages, whereas the availability of tetrahydrobiopterin was not a limiting factor in NO production after PD 98059. Our results indicate that Erk1/2 activation mediates up-regulation but is not essential for LPS-induced iNOS expression.

Regulation of nitric oxide production in cultured human T84 intestinal epithelial cells by nuclear factor-kappa B-dependent induction of inducible nitric oxide synthase after exposure to bacterial endotoxin.

BACKGROUND: Intestinal epithelium is consistently in contact with lipopolysaccharide (LPS) produced by intraluminal microbes. LPS induces nitric oxide production in many rodent cells, but in human cells it is very differently regulated. AIM: To test the hypothesis that exposure to LPS up-regulates nitric oxide synthesis in human intestinal epithelium. METHODS AND RESULTS: LPS induced nitric oxide synthesis in T84 cells in a time- and dose-dependent manner whereas detectable amounts of peroxynitrite were not produced. A novel selective inducible nitric oxide synthase (iNOS) inhibitor 1400 W potently inhibited LPS-induced nitric oxide synthesis in T84 cells while dexamethasone was relatively ineffective. Nitric oxide production was sensitive to cycloheximide, indicating that it was dependent on de novo protein synthesis. Nuclear factor-kappa B (NF-kappa B) inhibitor pyrrolidinedithiocarbamate abolished iNOS and nitric oxide production. Nitric oxide synthesis was also suppressed by genistein (tyrosine kinase inhibitor) and PD 98059 (p44/42 MAP kinase inhibitor) but enhanced by SB 203580 (p38 MAP kinase inhibitor). CONCLUSIONS: Intestinal epithelial cells express iNOS and produce nitric oxide in a nuclear factor-kappa B-dependent manner when exposed to LPS. The process is regulated by tyrosine kinases, and p44/42 and p38 MAP kinases. Because nitric oxide acts as an antimicrobial agent and immune modulator, these findings are implicated in the regulation of gut mucosal immunity.

Nitric oxide-releasing oxatriazole derivatives inhibit human lymphocyte proliferation by a cyclic GMP-independent mechanism.

Two novel nitric oxide (NO)-releasing oxatriazole derivatives, GEA 3162 and GEA 3175, and an earlier known NO donor, S-nitroso-N-acetylpenicillamine (SNAP), inhibited cell proliferation and enhanced cGMP production in a concentration-dependent manner in human lymphocytes activated by lectin mitogen concanavalin A (ConA). The possible mediator role of cGMP in the antiproliferative action of NO donors was tested by pharmacological means. An inhibitor of guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one, inhibited NO donor-induced cGMP production, whereas the antiproliferative action of NO donors remained unaltered. Phosphodiesterase inhibitors zaprinast and 3-isobutyl-1-methylxanthine potentiated and prolonged NO donor-induced increase in the concentrations of cGMP but did not enhance the antiproliferative action of NO donors. In addition, two analogs of cGMP, 8-bromo-cGMP and a more cell-permeable compound, 8-p-chlorophenylthio-cGMP, did not inhibit ConA-stimulated lymphocyte proliferation when used in concentrations of up to 300 microM. At millimolar concentrations, 8-bromo-cGMP had a moderate inhibitory action. These results suggest that nitric oxide-releasing oxatriazole derivatives inhibit proliferative responses in human lymphocytes by a cGMP-independent manner.

The subrenal capsule assay in selecting chemotherapy for ovarian cancer: a prospective randomized trial.

In order to find out whether the response rate and survival in epithelial ovarian cancer can be improved by aid of sensitivity testing with the subrenal capsule assay (SRCA), 196 patients with FIGO Stage II-IV epithelial ovarian cancer were randomized to be treated with either cyclophosphamide-doxorubicin-cisplatin (CAP) or SRCA-guided chemotherapy. The drug combinations tested with the SRCA were (1) cyclophosphamide-doxorubicin-carboplatin (CACAR), (2) CAP, (3) carboquone-methotrexate-tegafur (CQ-MTX-TEG), (4) cisplatin-etoposide-hexamethyl-melamine (P-VP-HXM), and (5) bleomycin-epirubicin-cisplatin (BEP). A total of 132 patients (CAP, 69; SRCA, 63) were eligible for efficacy analysis based on relaparotomy findings. The overall response rate was 59% in the CAP arm and 62% in the SRCA arm. In the SRCA arm, 16 patients were treated with CACAR, 24 with CAP, 10 with CQ-MTX-TEG, 11 with P-VP-HXM, and 2 with BEP. The response rate to CACAR was 63% and to SRCA-CAP was 75%. The number of complete responses was higher when CAP was given as guided by the assay than when given at random (14/24 vs 23/69; P = 0.03, Pearson chi 2). Survival curves as estimated by Kaplan-Meier method gave a median survival of 24 (SE = 4) months to the SRCA arm and 28 (SE = 5) for the CAP arm (P = 0.7; log-rank test). Because no survival benefit was achieved, the SRCA obviously needs further development before it can be routinely recommended in the choice of first-line chemotherapy for patients with ovarian cancer.

Aryl hydrocarbon hydroxylase activity in chemically induced and toremifene-treated mammary tumors in rats.

Aryl hydrocarbon hydroxylase (AHH) and NADPH-cytochrome P450 reductase (NCR) activity of microsomes from liver, lungs, uterus and mammary tumors in dimethylbenzanthracene-induced and toremifene-treated female Sprague-Dawley rats were studied. AHH and NCR activity in tumors and uteri was low compared with that in livers and lungs. The distribution of AHH in tumors was wide and skewed. It varied in different tumors of the same animal as widely as between different animals. The enzyme activity in tumors did not correlate with that in the liver, lungs or uterus of the same animal. Toremifene had no effect on AHH or NCR in tumor or liver, but it decreased them in lungs. Tumor AHH activity correlated with its overall growth rate and development stage. The results suggest that malignant transformation leading to the defect in growth regulation also confuses the complex regulatory system of AHH activity.

Expression of human placental cytochrome P450 aromatase (CYP19) cDNA in insect cells using a luciferase based baculovirus vector.

The cDNA encoding human placental cytochrome P450 aromatase (CYP19) was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression vector system. The recombinant protein product was characterized by Northern and Western blot analyses as well as by direct measurement of aromatase activity. The expressed enzyme proved to be both catalytically active in the presence of P450 reductase and immunologically reactive with polyclonal antibodies raised against human placental aromatase. The activity of aromatase increased 10% after the addition of 0.1 microgram/ml hemin chloride to the culture medium. However, the level of aromatase protein decreased considerably when the concentration of hemin chloride reached 10 micrograms/ml indicating that hemin chloride has toxic effects on the lepidopteran insect cell line. In conclusion, the baculovirus system is suitable for high level expression of functional human placental CYP19.

Cytostatic effect of interferon alpha on human bladder cancer cells in vitro. A flow cytometric study.

The cytostatic activity of interferon a-2b on human transitional cell cancer T24 cells was examined using bioluminescence assay. Interferon a in a concentration of 1 x 10(6) IU/ml had a remarkable cytostatic effect already in two-day cultures, and this effect increased still further when the cells were incubated for three and five days. In the flow cytometric study, the percentage of cells in the S-phase fraction (SPF) remained at the same level after three days of incubation with interferon a (43.6 +/- 2.7), but decreased significantly in the control cell population (25.6 +/- 1.5). If interferon a was washed away after two hours and incubation was continued for three days, the percentage of cells in the SPF was also significantly higher (43.6 +/- 3.3) than that in the control group of cells (P < 0.01).

Uptake of 2-fluoro-2-deoxy-D-[U-14C]-glucose during chemotherapy in murine Lewis lung tumor.

Mice bearing intramuscular Lewis lung tumor were treated with BCNU and doxorubicin (ADM) to study chemotherapy-induced changes in the uptake of 2-fluoro-2-deoxy-[U-14C]glucose (FDG). A decreased FDG uptake, tumor regression and a diminished proportion of aneuploid versus diploid cells as evaluated by DNA flow cytometry were seen after treatment with BCNU but not with ADM; HPLC indicated that most of the 14C activity in tumors was from FDG6-phosphate. The results suggest that changes in FDG uptake reflect the effectiveness of antitumor therapy. FDG may be valuable in follow-up studies of cancer treatment.

Cytostatic effect of different strains of Bacillus Calmette-Guérin on human bladder cancer cells in vitro alone and in combination with mitomycin C and interferon-alpha.

The cytostatic activity of five Bacillus Calmette-Guérin (BCG) strains (Pasteur, Evans, Tice, RIVM and Connaught) on human transitional cell cancer T24 cells was examined. A striking effect was noted even in 2-day cultures, and the effect was more pronounced when the cells were incubated for 5 days with different BCG strains alone. The concentrations needed were about the same as those used in clinical practice (10(9) colony-forming units of Pasteur strain in 100 ml buffered saline solution). Combination with mitomycin C or interferon-alpha-2b potentiated the cytostatic effect. A slight difference in cytostatic activity between different BCG strains was found.

Extracellular production of cloned alpha-amylase by Escherichia coli.

Overexpression of Bacillus stearothermophilus gene coding for thermostable alpha-amylase in Escherichia coli was shown to cause outer-membrane damage leading to extracellular location of periplasmic proteins. Prolonged high expression of the alpha-amylase gene under lacZpo control eventually also lysed cells. Surprisingly, expression controlled by the pL promoter of phage lambda allowed specific release of periplasmic proteins into the growth medium without total cell lysis. Accumulation of alpha-amylase in the growth medium continued for at least 24 h under lambda pL control, whereas beta-lactamase activity ceased to increase beyond the exponential growth phase. The extent of outer membrane damage caused by alpha-amylase expression was monitored by following growth kinetics in the presence of lysozyme and by electron microscopy of the cells. Supplementing growth medium with Mg2+ restored the normal growth kinetics. It is suggested that periplasmic protein release caused by alpha-amylase overexpression is a stress response of the cell. A role for induced autolytic activity of the cell as a final effector of protein release is also proposed.