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Inhibition of proliferation and induction of differentiation of human and mouse myeloid leukemia cells by new ethyleneglycol-type nonphosphorus alkyl ether lipids.

A variety of ethyleneglycol-type nonphosphorus alkyl ether lipids, ether derivatives of diethyleneglycol in which the two hydroxyl groups were substituted with long chain alkyl and quaternary ammonioalkyl groups, were synthesized and their effects on proliferation and differentiation of cultured human (HL-60) and mouse (M1) myeloid leukemia cells were studied. Incubation with these compounds inhibited the cellular proliferation, and the cells differentiated into morphologically and functionally mature granulocytes. Of the compounds tested, 1-[2-[2-(octadecyloxy)ethoxy]ethoxy]-butylpyridinium mesylate (EG-6) was the most effective in inducing differentiation of HL-60 cells. Almost maximal induction of differentiation and inhibition of growth of HL-60 cells on day 6 were observed when the cells were treated with EG-6 for 1 day and then cultured without EG-6 for a further 5 days. The inhibitory effect of EG-6 on the leukemic cells was over 100 times more than that of 2-[2-(dodecyloxy)ethoxy]ethyl 2-pyridinioethyl phosphate, a potent antileukemic ether phospholipid.

Animals↗

Phosphate determination by enzymatic colorimetric assay.

A direct colorimetric assay for inorganic phosphate in serum is described. The system is based on utilization of the enzymes, purine-nucleoside phosphorylase and xanthine oxidase, to generate superoxide ions. The superoxide is measured in the presence of an electron mediator compound with 3-(4',5'-dimethyl-2-thiazolyl)-2,4-diphenyl-2H-tetrazolium bromide as the chromogen. The high absorbance of this chromogen between 550 and 660 nm affords useful results with a sample/reagent volume ratio as low as 1:100. A single working reagent is used, and the reaction is complete in 15 min at room temperature. The standard curve is linear for inorganic phosphate concentrations as high as 4.9 mmol/liter. Analytical recovery of phosphate in human sera averages 100%. Within-run precision study gives CV less than or equal to 1.0%. The results of this method compare closely (r greater than 0.99) with those obtained by the semidine method (recommended standard). The method lends itself to automation.

Colorimetry↗

Topographic estimations by component spectroanalysis of two formazans of nitroblue tetrazolium in tissue sections.

A component-spectroanalysis technique was used to study the multicolor properties of histochemically stained tissue sections. We developed a method that makes it possible to obtain separately both the spectral patterns and spatial distributions of different color components in tissue sections. To illustrate the application of this technique, we examined the extinction spectrum of reduced nitroblue tetrazolium (NBT), which is used for the detection of dehydrogenase activity. Upon the reduction of NBT, mono- and diformazans are formed, and these exhibit over-lapping extinction spectra. When succinate dehydrogenase (SDH) activity in rat liver lobules was examined using NBT, monoformazan was found to be present at higher concentrations than diformazan and to have a uniform distribution, whereas the concentration of diformazan increased with a steep gradient between the center and periphery of lobules. In rat skeletal muscle fibers, diformazan was present at higher concentrations than monoformazan. The level of SDH activity was topographically represented by the hydrogen concentration calculated from the concentrations of the two formazans. This method is effective for separating multiple components such as mono- and diformazans in histochemical reactions.

Animals↗

Histochemical demonstration of non-specific esterases and non-specific acid phosphatases using menadiol substrates.

Although many synthetic substrates and methods are available for the histochemical detection of non-specific esterases and non-specific acid phosphatase, there are still further possibilities to investigate these hydrolases histochemically. This was shown for menadiol diacetate and menadiol diphosphate using tetrazolium salt, simultaneous azo-dye as well as metal salt methods in many rat tissues. In comparison, the azo-dye procedure with various Fast salts or hexazonium Pararosaniline or New Fuchsin delivered less satisfactory results; precisely localized stain in sufficient amounts was obtained for non-specific esterases using nitro BT, tetranitro BT or benzothiazolystyrylphthalhydrazidyl tetrazolium (BSPT) and for non-specific acid phosphatase with BSPT in the tetrazolium salt method or using cerium ions for phosphate trapping in the diaminobenzidine-nickel-hydrogen peroxide procedure.

Acid Phosphatase↗

Cyclosporin G inhibits proliferation of A431 cells in a dose- and time-dependent manner comparable to cyclosporin A.

Cyclosporin A (CyA), a fungal metabolite with potent immunosuppressive activity and an antiproliferative effect on epithelial cells, i.e. normal and transformed keratinocytes, is currently proposed in the treatment of psoriasis, where its use is limited mainly by possible nephrotoxicity and/or hepatotoxicity. Numerous analogs of CyA have been produced and studied. The most promising of these is the immunosuppressive analog cyclosporin G (CyG), in which norvaline is substituted for alpha-aminobutyric acid at the 2 position. This would maintain strong immunological activity, with reduced to absent nephrotoxic and hepatotoxic effects. The authors compared the antiproliferative effect of CyG and CyA on the epidermoid carcinoma cell line A431 in vitro, performing the MTT-microculture tetrazolium colorimetric assay based on the ability of viable cells to reduce the MTT compound to a blue formazan product. Subconfluent A431 cells were incubated with CyA or CyG or solvent only, for 24, 48, 72 or 96 h at concentrations of in vivo relevance (0.3, 0.6, 1.25, 2.5, 5, 7.5, 10 micrograms/ml). CyA and CyG showed similar antiproliferative effects, in low-serum-containing media in a dose- and time-dependent manner. After 24 h of incubation, the inhibition of the growth rate was irrelevant. A striking inhibition of the growth rate at the higher concentrations of the drugs (7.5 and 10 micrograms/ml) at 72 and 96 h of incubation was evident. Therefore CyG has been demonstrated to exercise an antiproliferative effect on the A431 cell line. These data suggest possible use for CyG in the treatment of immune-mediated disease, particularly in the treatment of dermatologic diseases characterized by epidermal hyperplasia and/or keratinocyte hyperproliferation.

Cell Division↗

Newer formazans and tetrazolium indoles as potential CNS-active agents.

Some new 2-aryl-5-(3'-indole)-3'-substituted phenyl tetrazolium bromide/iodide salts (IV) were synthesized by the oxidation of 1-aryl-3-(3'-indole)-5-substituted phenyl fromazans (III). These compounds III and IV were evaluated for their monoamine oxidase (MAO) inhibitory activity in vitro and various CNS activities in vivo. Several compounds exhibited promising CNS activities, III-b being the most active member of the present series showing marked antidepressant and MAO inhibitory activities and having low toxicity.

Animals↗

Triphenyltin acetate (TPTA)-induced cytotoxicity to mouse thymocytes.

To perform a better investigation of the toxic activity of triphenyltin acetate (TPTA) already described in vivo, primary cultures of murine thymocytes were incubated for 2 to 32 h with graded amounts (0.5-8 microM) of the triorganotin compound. The cytotoxic activity has been evaluated with the Trypan blue dye exclusion test, the 3-(4,5-dimethyl-thiazol-2-yl)-2,5 diphenyl-tetrazolium bromide (MTT) assay for cell survival and the cellular release of lactate dehydrogenase. Following 2 h of incubation with TPTA, a dose-dependent reduction (P < 0.05) of cellular viability occurred and marked increases (P < 0.05) of the MTT cytotoxic index and of lactate dehydrogenase leakage were also observed. These findings indicate that TPTA is as cytotoxic to mice thymocytes, as other triphenyltin derivatives are to other species.

Animals↗

Inhibition of ferrous iron induced oxidation of arachidonic acid by indomethacin.

The molecular mechanism by which indomethacin exerts its inhibitory effects on the prostaglandin endoperoxide synthetase enzyme is unknown. In the present study we have explored the possibility that indomethacin might interact with Fe++ in the enzyme to produce its inhibitory effect. For this study we made use of the recent discovery that Fe++ alone can oxidize arachidonic acid, and the interaction of this fatty acid with the metal can be detected by following reduction of nitroblue tetrazolium (NBT) or by conversion of the Fe++ to Fe+++. Indomethacin markedly inhibited NBT reduction in the presence of arachidonic acid and Fe++ when the indomethacin had been preincubated with the Fe++. Indomethacin also inhibited the conversion of Fe++ to Fe+++ by arachidonic acid. Results obtained by varying the concentrations of indomethacin and arachidonic acid and measuring inhibition of the conversion of Fe++ to Fe+++ by the indomethacin are consistent with a one to one complex forming between indomethacin and Fe++. The complex between indomethacin and Fe++ separates on prolonged incubation of the complex with arachidonic acid. The nature of the binding is suggested by a molecular model. Our results suggest that indomethacin may act to inhibit the prostaglandin endoperoxide synthetase enzyme by complexing Fe++ in the enzyme. Ibuprofen and tolmetin, two other prostaglandin synthetase inhibitors, also inhibit the interaction of Fe++ with arachidonic acid suggesting this may be a general mechanism for this type of drug.

Arachidonic Acids↗

Nature and significance of the electron-dense bodies of the endospores of rhinosporidium seeberi: their reactions with MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) and TMRE (tetramethyl-rhodamine ethyl ester).

The most conspicuous internal structures of the endospore of Rhinosporidium seeberi are the 10-16 spherical, 1.0-1.5-microm bodies that have been termed electron-dense bodies (EDB) or lipid bodies (LB); some authors have regarded them as nutritive stores of lipid or protein while others have regarded them as DNA-containing, ultimate generative units of R. seeberi. The literature is reviewed as supporting either view. We report, for the first time, (i) reactions of the endospores with the salt MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) and (ii) ultrastructural appearances; and suggest that both views on the nature of spherical bodies are valid (i.e. the endospore contains both EDB, and lipid or protein bodies). Well-marked reduction of the MTT with the production of deep-purple staining was seen in a proportion of the spherical bodies, probably the EDB, suggesting that they are actively metabolizing, viable elements with dehydrogenase activity, and that these bodies are the thick-walled electron-dense bodies described as EDB and visualized in the transmission electron microphotograph illustrated in this paper. The spherical bodies showed fluorescent labeling with acridine orange and with ethidium bromide supporting the idea that they contained nucleic acids. TMRE (tetramethyl rhodamine ethyl ester), a mitochondrion-specific dye, also labeled the intra-endosporial spherical bodies. Other bodies (LB) of a similar size that were MTT-non-reducing, electron lucent, and have no organized structure, are probably the lipid or protein containing, inert, nutritive storage bodies suggested by previous authors.

Acridine Orange↗

Inhibition of xanthine oxidase by pterins.

The effect of a panel of pterins on xanthine oxidase was investigated by measuring formation of urate from xanthine as well as formazan production from nitroblue tetrazolium. The pterin derivatives, depending on their chemical structure, decreased urate as well as formazan generation: 200 microM neopterin and biopterin suppressed urate formation (90% from baseline) and formazan production (80% from baseline) as well. Their reduced forms, 7,8-dihydroneopterin and 5,6,7,8-tetrahydrobiopterin, showed a lesser but still strongly diminishing influence (40% from baseline). Another oxidized pterin namely leukopterin showed only a weak inhibitory effect. Xanthopterin, a known substrate of xanthine oxidase, had a strong effect on urate formation (80% inhibition), but a lesser effect on formazan production (30% reduction). When iron-(III)-EDTA complex was added to the reaction mixture all the effects were more pronounced. Superoxide dismutase, which removes superoxide anion by dismutation into oxygen, decreased formazan production in addition to pterin derivatives and had a small but enhancing effect on urate formation. Also the reductant N-acetylcysteine had an additive effect to pterins to diminish formazan production in a dose-dependent way. The results of our study suggest that depending on their chemical structure pterins reduce superoxide anion generation by xanthine oxidase.

Acetylcysteine↗

Modulatory properties of various natural chemopreventive agents on the activation of NF-kappaB signaling pathway.

PURPOSE: To study and compare effects of selected natural chemopreventive agents on the transcription activation of nuclear factor-kappa B (NF-kappaB) in human HT-29 colon cancer cells. METHODS: The natural chemopreventive compounds isothiocyanates (ITCs) found in cruciferous vegetables, flavonoids found in green tea, resveratrol (RES) and procyanidin dimers found in red wine, and curcumin (CUR) found in turmeric curry food were examined in this study. HT-29 cells were stably transfected with NF-kappaB luciferase construct, and stable clones were selected. One of the clones, HT-29 N9 cells, was selected and treated with various concentrations of the natural chemopreventive agents and subsequently challenged with NF-kappaB stimulator lipopolysaccharide (LPS), and the luciferase activities were measured. Western blot analysis of phosphorylated IkappaBalpha was performed after treatments with the natural chemopreventive agents. The effects of these agents on cell viability and apoptosis were also evaluated by a nonradioactive cell proliferation MTS assay [3-(4,5-dimethylthiazol-2-yl)-5-(3-arboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]. Trypan blue staining, and caspase assay. RESULTS: Treatments with the natural chemopreventive compounds resulted in different responses in the NF-kappaB-luciferase assay. ITCs such as phenethyl isothiocyanate (PEITC), sulforaphane (SUL), allyl isothiocyanate (AITC), and curcumin (CUR) strongly inhibited LPS-induced NF-kappaB-luciferase activations, whereas RES increased activation at lower dose, but inhibited activation at higher dose, and tea flavonoids and procyanidin dimers had little or no effects. ITCs, CUR, (-)-epigallocatechin-3-gallate (EGCG), and RES reduced LPS-induced IkappaBalpha phosphorylation. Furthermore, in the MTS assay, PEITC, SUL, and CUR also potently inhibited cell growth. Caspase-3 activity was induced by chemopreventive compounds, however, the kinetics of caspase-3 activation varied between these compounds within the 48-h time period. CONCLUSIONS: These results suggest that natural chemopreventive agents have differential biological functions on the signal transduction pathways in the colon and/or colon cancer.

Anticarcinogenic Agents↗

Interaction of alpha-phenyl-N-tert-butyl nitrone and alternative electron acceptors with complex I indicates a substrate reduction site upstream from the rotenone binding site.

Mitochondrial complexes I, II, and III were studied in isolated brain mitochondrial preparations with the goal of determining their relative abilities to reduce O2 to hydrogen peroxide (H2O2) or to reduce the alternative electron acceptors nitroblue tetrazolium (NBT) and diphenyliodonium (DPI). Complex I and II stimulation caused H2O2 formation and reduced NBT and DPI as indicated by dichlorodihydrofluorescein oxidation, nitroformazan precipitation, and DPI-mediated enzyme inactivation. The O2 consumption rate was more rapid under complex II (succinate) stimulation than under complex I (NADH) stimulation. In contrast, H2O2 generation and NBT and DPI reduction kinetics were favored by NADH addition but were virtually unobservable during succinate-linked respiration. NADH oxidation was strongly suppressed by rotenone, but NADH-coupled H2O2 flux was accelerated by rotenone. Alpha-phenyl-N-tert-butyl nitrone (PBN), a compound documented to inhibit oxidative stress in models of stroke, sepsis, and parkinsonism, partially inhibited complex I-stimulated H2O2 flux and NBT reduction and also protected complex I from DPI-mediated inactivation while trapping the phenyl radical product of DPI reduction. The results suggest that complex I may be the principal source of brain mitochondrial H2O2 synthesis, possessing an "electron leak" site upstream from the rotenone binding site (i.e., on the NADH side of the enzyme). The inhibition of H2O2 production by PBN suggests a novel explanation for the broad-spectrum antioxidant and antiinflammatory activity of this nitrone spin trap.

Animals↗

O6-alkylguanine-DNA alkyltransferase activity of human malignant glioma and its clinical implications.

Activity of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) is an important determinant of responsiveness of tumor cells to chloroethylnitrosoureas (CENUs), representative chemotherapeutic agents for primary malignant gliomas. In order to assess the real states of this repair protein in human malignant gliomas, we assayed AGT activity in surgically extirpated 42 malignant glioma samples and studied the distribution of the activity under certain clinical conditions. There were wide variations in AGT activity between individuals. No significant difference in AGT activity on average was seen either between glioblastoma and anaplastic astrocytoma, nor between primary and recurrent tumors. Among 42 malignant gliomas, 7 samples (16.7%) had low AGT activity less than 0.1 pmoles/mg protein. In the case of glioblastoma, tumors possessing higher AGT activity tended to be less responsive to post-operation remission-induction therapy including CENUs. The result of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) chemosensitivity assay by using the corresponding surgical specimens suggested a close relationship between cellular resistance to CENUs and AGT activity. It was found to be unlikely that a short term administration of CENUs had a significant effect on AGT activity of brain tumors in human body. We could detect a bit of definite evidences of the relevance of AGT to resistance to CENUs and need to conduct further investigations for other resistance factors.

Astrocytoma↗

Antioxidative activity of synthetic melanins. Cardiolipin liposome model.

The inhibiting effect of melanin synthesized from dihydroxyphenylalanine (DOPA), dopamine, adrenaline and adrenolutin on the ultraviolet- or the Fe(2+)-ascorbic acid-induced peroxidation of cardiolipin liposomes has been studied. All these melanins are able to inhibit both the ultraviolet- and the Fe(2+)-ascorbic acid-induced lipid peroxidation. Antioxidative activity of melanins enhances in the order: dopamine-melanin less than melanin synthesized from dopamine in the presence of Cu(2+) less than DOPA--melanin less than melanin synthesized from adrenaline in the presence of Cu(2+) approximately equal to adrenolutin-melanin, and correlates with their ability to scavenge superoxide anion radical. The optical screening effect of the investigated melanins in the inhibition of lipid peroxidation was not higher than 15% for the most active melanins.

Antioxidants↗