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Cell-based screening approach for antitumor drug leads which exploits sensitivity differences between normal and cancer cells: identification of two novel cell-cycle inhibitors.

A cell-based in vitro screening approach for identification of antitumor drug leads that exploits the differential sensitivity between normal and cancer cells was developed. It is a three-step, high-throughput screen for antiproliferative and/or cytotoxic activity measured by a 7 day MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromidel assay using small panels of proliferating primary human cells and established cancer cell lines. Proof-of-concept experiments successfully identified 11 known cancer drugs randomly mixed with 5000 test compounds. Application of this screening approach to a library of 110000 compounds allowed for the identification of several novel chemical classes of compounds active against an expanded panel of cancer cell lines in vitro. Two of the compounds representing novel mitotic inhibitors with in vivo potency against established breast cancer xenografts (MDA-MB-435) are reported here.

Antineoplastic Agents↗

Simple and rapid portable chromatographic method for separation and detection of phenylmercuric acetate in seeds and water.

Phenylmercuric acetate can be detected by horse liver acetone powder succinate dehydrogenase inhibition, using a mixture of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT), sodium succinate, and N-methylphenazonium methosulfate as the chromogenic reagent. The simple cleanup involves extraction of phenylmercuric acetate in chloroform and concentration by evaporation. In the extract, the compounds in seeds or water could be separated and identified by paper chromatography in the field or laboratory at microgram levels with an acetone-water (70 + 30) solvent system.

Chromatography, Paper↗

Evaluation of anti-herpetic and antioxidant activities, and cytotoxic and genotoxic effects of synthetic alkyl-esters of gallic acid.

The n-alkyl esters of gallic acid (CAS 13857-8) have a diverse range of uses as antioxidants in food, cosmetics and pharmaceutical industries. Pharmaceutical studies performed with these compounds have found that they have many therapeutic potentialities including anti-cancer, antiviral and antimicrobial properties. However, more interest has been devoted to their antioxidant activity due to the ability to scavenge and reduce reactive oxygen species (ROS) formation. In this study, gallic acid and 14 different alkyl gallates were tested. The cytotoxicity and anti-herpetic (HSV-1, KOS and 29-R strains) activity were studied by using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) colorimetric assay and the cell viability by using the Trypan blue dye exclusion method. The genotoxicity was studied by the Comet assay and the antioxidant activity by using the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging and microsomal lipid peroxidation-inhibiting activities. The results showed that all the tested compounds have anti-herpetic activity at non cytotoxic concentrations with selectivity indices (SI = CC50/EC50) varying from 0.89 to 18.34, depending on the used HSV-1 strain. It was observed that all tested alkyl gallates showed some degree of genotoxicity, at the tested concentrations, except cetyl gallate, at 256.60 micromol/L (p <0.05, t-Student test), probably induced by ROS released by infected cells and/or by the alkyl gallates that were not antioxidants, at the tested concentrations, in which they demonstrated anti-herpetic activity. The hydroxyl groups can induce DNA damage due interactions with some metal ions, which are naturally present in the culture medium supplemented with fetal bovine serum, probably explaining the genotoxicity detected. However, the obtained results showed considerable antioxidant activity at smaller concentrations, when compared to quercetin which is considered as a reference drug due to its already described antioxidant potential: DPPH radical scavenging activity with IC50 values varying from 17 to 31 micromol/L; and microsomal lipid peroxidation-inhibiting activity with IC50 values varying from 21 to 59 micromol/L. It was observed that the presence of hydroxyl groups in these molecules is important for their pharmacological profile, but the length of the lateral carbonic chain does not have considerable influence.

Animals↗

Cytotoxicity and cellular accumulation of a new cis-diammineplatinum (II) complex containing procaine in murine L1210 cells sensitive and resistant to cis-diamminedichloroplatinum (II).

The emergence of drug resistance during tumor chemotherapy is one of the main problems associated with cancer treatment, particularly with cisplatin (cis-DDP). In the hope of overcoming this problem, various cis-DDP-derived compounds have been synthesized, and their pharmacological activity was compared with that of cis-DDP. In this paper we report on studies on the cytotoxic activity induced by cis-diamminechloro-[2-(diethylamino)ethyl-4-aminobenzoate, N4]- chlorideplatinum(II) monohydrochloride monohydrate (DPR), a new complex of platinum containing procaine. All experiments were carried out on murine leukemic cells, which were either sensitive (L1210) or resistant (L1210/DDP) to cis-DDP. A tetrazolium dye (MTT) assay conducted 5 days after a 2-h exposure of cells to both drugs was utilized to determine the resistance factor (RF) of L1210/DDP cells as compared with the sensitive wild-type cells. Drug accumulation and efflux, together with the amount of platinum bound to DNA, were also investigated. The activity of DPR on sensitive cells was not significantly different from that of cis-DDP. Conversely, DPR was 4.3 times more effective than cis-DDP on resistant cells. A decreased drug accumulation is one of the mechanisms of resistance to cis-DDP of L1210/DDP cells. However, DPR accumulation was not significantly different in sensitive and resistant L1210 cells. Under culture conditions that yielded similar intracellular platinum concentrations, treatment with DPR produced significantly greater DNA platination than did treatment with cis-DDP in both cell lines. No difference in efflux was observed between L1210 and L1210/DDP cells exposed to either cis-DDP or DPR. Our results show that in parental cells, DPR is as potent as cis-DDP on a molar basis, and it is also minimally cross-resistant with cis-DDP in L1210/DDP cells. A direct implication of our results is that DPR could be useful in those human tumors showing a mechanism of resistance similar to that of L1210/DDP cells.

Animals↗

Toxicity of camphorated phenol and camphorated parachlorophenol in dental pulp cell culture.

The toxicity of phenol, parachlorophenol, camphorated phenol, camphorated parachlorophenol, and camphor was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay on established rat dental pulp cells (RPC-C2A). RPC-C2A cells at the confluent stage were incubated for 24 h in an experimental medium containing each compound at different concentrations. All tested drugs showed cytotoxicity in the MTT assay in a concentration-dependent manner. It is believed that camphor is a vehicle, and it reduces the toxicity of phenol and parachlorophenol. However, camphor itself showed cytotoxicity, and the addition of camphor increased the toxicity of phenol and parachlorophenol, reconfirming the cytotoxicity of these classical antiseptics.

Analysis of Variance↗

Microplate Alamar blue assay for Staphylococcus epidermidis biofilm susceptibility testing.

Biofilms are at the root of many infections largely because they are much more antibiotic resistant than their planktonic counterparts. Antibiotics that target the biofilm phenotype are desperately needed, but there is still no standard method to assess biofilm drug susceptibility. Staphylococcus epidermidis ATCC 35984 biofilms treated with eight different approved antibiotics and five different experimental compounds were exposed to the oxidation reduction indicator Alamar blue for 60 min, and reduction relative to untreated controls was determined visually and spectrophotometrically. The minimum biofilm inhibitory concentration was defined as < or = 50% reduction and a purplish well 60 min after the addition of Alamar blue. All of the approved antibiotics had biofilm MICs (MBICs) of >512 microg/ml (most >4,096 microg/ml), and four of the experimental compounds had MBICs of < or = 128 microg/ml. The experimental aaptamine derivative hystatin 3 was used to correlate Alamar blue reduction with 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction and viable counts (CFU/ml) for S. epidermidis ATCC 35984, ATCC 12228, and two clinical isolates. For all four strains, Alamar blue results correlated well with XTT (r = 0.83 to 0.97) and with CFU/ml results (r = 0.85 to 0.94). Alamar blue's stability and lack of toxicity allowed CFU/ml to be determined from the same wells as Alamar blue absorbances. If the described method of microplate Alamar blue biofilm susceptibility testing, which is simple, reproducible, cost-effective, nontoxic, and amenable to high throughput, is applicable to other important biofilm forming species, it should greatly facilitate the discovery of biofilm specific agents.

Anti-Bacterial Agents↗

Effect of butylated hydroxytoluene on bilineage differentiation of the human HL-60 myeloblastic leukemia cell.

Butylated hydroxytoluene (BHT), which has both antioxidant and membrane active properties, has been reported to affect cellular differentiation. We studied its effect on the bipotent lineage differentiation of the important HL-60 human myeloblastic leukemia cell line using reduction of nitroblue tetrazolium, cell cycle analysis, population growth rate, monoclonal antibodies, and morphology. BHT markedly accelerated retinoic acid-induced myelocytic differentiation and dihydroxyvitamin D3-induced monocytic differentiation in a concentration and time-dependent manner. Butylated hydroxyanisole (BHA) had a comparable effect. Preincubation with the compounds was not necessary to evoke the acceleration Other antioxidants and inhibitors of eicosanoid synthesis were inactive. We conclude that the important food preservatives BHT and BHA accelerate the kinetics of terminal differentiation of human leukemia and that this effect is likely due at least in part to their membrane active properties.

Antigens, Differentiation, Myelomonocytic↗

Colorimetric lactate dehydrogenase (LDH) assay for evaluation of antiviral activity against bovine viral diarrhoea virus (BVDV) in vitro.

A rapid and sensitive screening assay has been established for in vitro evaluation of antiviral compounds against bovine viral diarrhoea virus (BVDV), which is widely used as a surrogate for hepatitis C virus (HCV). The procedure is based on photospectrometrical assessment for the viability of virus-infected cells via extracellular leakage of lactic dehydrogenase (LDH). The level of LDH in culture supernatants of BVDV-infected Madin-Darby bovine kidney (MDBK) cells was significantly higher than those of mock-infected MDBK cells. Under optimized assay conditions, the LDH level was found to correlate well with the degree of viral replication. When the 50% effective concentrations (EC50s) of ribavirin, cyclosporine A and human interferon-alpha for BVDV replication were determined by the established LDH method and compared with those obtained by a conventional tetrazolium colorimetric (MTT) method, there was a complete correlation in EC50s between the two methods. Furthermore, a much higher ratio of background activity (noise) to sample activity (signal) could be achieved with the LDH method than with the MTT method, indicating that the present LDH assay permits a sensitive, rapid and reliable screening of compounds for their anti-BVDV activity and may be useful for the discovery of novel anti-HCV agents.

Animals↗

Paradoxical effects of adenosine on neutrophil chemotaxis.

Chemotaxis of rabbit neutrophils is most sensitive to inhibition by 3-deazaadenosine, followed by 3-deaza-(+/-)aristeromycin, 3-deaza-(+/-)aristeromycinylhomocysteine, 3-deazaadenosylhomocysteine, and adenosylhomocysteine, in that order. Although adenosine by itself had no effect on the chemotaxis of neutrophils, it essentially abolished the inhibitory effects of 3-deaza-adenosine on chemotaxis and the reduction of nitroblue tetrazolium. Paradoxically, adenosine enhanced the inhibition of chemotaxis by 3-deazaadenosylhomocysteine slightly and that of 3-deaza-(+/-)aristeromycin significantly. Adenosine alone unexpectedly inhibited phospholipid methylation to the same extent as 3-deazaadenosine, and reduced protein carboxymethylation to a lesser degree. The inhibition of these two methylation reactions by 3-deazaadenosine was, however, not substantially altered in the presence of adenosine. Drastic changes in the ratio of adenosylmethionine/nucleosidylhomocysteine were observed in the presence of adenosine, 3-deazaadenosine, 3-deaza-(+/-)aristeromycin, or of adenosine in combination with each of the latter compounds. There was no significant effect on the binding of chemotactic peptide to receptors, or on the ratio of ATP/ADP in cells treated by the analogs. These results suggest that the inhibition of methylation reactions per se is not enough to account for the inhibition of both chemotaxis and the reduction of nitroblue tetrazolium by neutrophils.

Adenosine↗

Antioxidant activity of Hypericum androsaemum infusion: scavenging activity against superoxide radical, hydroxyl radical and hypochlorous acid.

Hypericum androsaemum is a medicinal plant species containing many polyphenolic compounds, namely flavonoids and phenolic acids. Since polyphenolic compounds have high antioxidant potential, the ability of H. androsaemum infusion to act as a scavenger of reactive oxygen species (superoxide radical, hydroxyl radical and hypochlorous acid) was investigated. Superoxide radical was generated by the xanthine/xanthine oxidase and phenazine methosulphate/NADH systems. The infusion-mediated prevention of nitroblue tetrazolium reduction by the superoxide radical was used as the measured endpoint. Hydroxyl radical was generated by the Fe3+-EDTA/ascorbate Fenton system, and assayed by evaluating deoxyribose degradation using the thiobarbituric acid method. Hypochlorous acid scavenging activity was tested by measuring the inhibition of hypochlorous acid-induced 5-thio-2-nitrobenzoic acid oxidation to 5,5'-dithiobis(2-nitrobenzoic acid). The tested infusion mainly exhibited a potent scavenging effect on superoxide radicals (although a noncompetitive inhibitory effect on xanthine oxidase was also observed). The infusion also acted as a moderate scavenger of hydroxyl radicals and hypochlorous acid. A phytochemical study of the infusion was also undertaken, and nine phenolic compounds were identified.

Antioxidants↗

Rapid decline in folylpolyglutamate synthetase activity and gene expression during maturation of HL-60 cells. Nature of the effect, impact on folate compound polyglutamate pools, and evidence for programmed down-regulation during maturation.

These studies in HL-60 cells examined the regulation of folylpolyglutamate synthetase (FPGS) activity at the level of gene expression during terminal maturation. Following addition of 210 mM Me2SO to cultures of HL-60 cells at a concentration that induces maturation of 85-90% of the cells, FPGS activity, but not folylpolyglutamate hydrolase (FPGH) activity, was reduced 2-7-fold within 1-5 days. The initial decline in FPGS activity preceded any effect of Me2SO on rate of growth and the increase in appearance of nitro blue tetrazolium-positive cells, a marker of cellular maturation, and the decrease after 5 days of exposure to Me2SO was solely accounted for by a 7-fold decrease in value for Vmax. The same time and concentration dependence for Me2SO was shown for the decline in FPGS activity, increase in nitro blue tetrazolium-positive cells, and decline in the level of a 2.1-kilobase FPGS mRNA during exposure to this inducer. This decline in FPGS mRNA was reversible when Me2SO was removed from the culture medium but only until that time when an appreciable number of cells were committed to terminal maturation. Following growth of HL-60 cells with [3H]MTX, used as a model folate compound, a large reduction in its intracellular polyglutamate pools was shown during maturation which quantitatively reflected the decline in FPGS activity as well as folate transport inward (Sirotnak, F.M., Jacobson, D.M., and Yang, C-H. (1986) J. Biol. Chem. 261, 11150-11156). Other data showed that folate status or obviation of the folate requirement during growth of these cells strongly influenced the rapidity of the onset of maturation following exposure to inducer. Overall, these results show that FPGS activity in HL-60 cells is a marker for proliferative capacity and that the underlying basis for the decline in FPGS activity during maturation is altered cognate gene expression which is manifested as early reversible and late irreversible phases. They also suggest that the coordinate reduction observed in folate transport, FPGS activity, dihydrofolate reductase, and probably other folate related enzymes by limiting macromolecular biosynthesis may be early programmed events in the maturation process that influence the switch from proliferation to senescence in these cells.

Blotting, Northern↗

Behavioral effects of photothrombotic ischemic cortical injury in aged rats treated with the sedative-hypnotic GABAergic drug zopiclone.

Sedative-hypnotic drugs commonly used in the elderly may affect functional recovery following cerebrovascular events. Previous research has shown that prolonged exposure to diazepam can interfere with recovery of function and exaggerate tissue loss after brain injury. The present study evaluated the effect of zopiclone, a widely used hypnotic drug, on functional and histological outcome after cortical photothrombosis in aged rats, which might be particularly vulnerable to brain insults and inhibitory sedative-hypnotic drugs. Aged Wistar rats were treated with zopiclone at a dose of 3 mg/kg (i.p., once a day) beginning 4 days before ischemia induction and continuing for 23 days. Sensorimotor recovery was assessed by a new ledged beam-walking test and spatial learning by the Morris water-maze. After a 7-day washout period all rats were administered a single dose of zopiclone (3 mg/kg, i.p.) and retested. Infarct volumes were measured from nitroblue tetrazolium-stained sections at the end of the experiment. Beam-walking data showed that ischemic rats treated with zopiclone were not more impaired than untreated rats. Indeed, they showed fewer faults with the impaired hindlimb than ischemic controls on post-operative day 16. Water-maze performance was not affected by zopiclone. After the washout period a single dose of zopiclone did not worsen forelimb or hindlimb function, but seemed to improve performance in the water-maze test. Cortical infarct volumes were similar in ischemic controls and ischemic rats treated with zopiclone. In conclusion, zopiclone was not detrimental and even seemed to improve behavioral outcome without affecting ischemic damage in aged rats subjected to cortical photothrombosis.

Aging↗

Structure-staining relationships in histochemistry and biological staining. Part 3. Some comments on the intentional and artifactual staining of lipids.

The choice, basis and calculation of certain numerical coefficients for describing chemical structures of such staining reagents as dyes, enzyme substrates and visualising agents as described. The general value of electric charge, conjugated bond number, Hansch pi values, and molecular or ionic weights is emphasised. Hansch pi values as indicators of hydrophobic character, are then used to analyse reagents giving rise to the staining of neutral lipids. Dyestuffs used to stain lipids all have high Hansch pi values (greater than or equal to +1.0), with superior stains, e.g. Sudan Black B and Oil Red O, having values of greater than or equal to +7.0. In keeping with this, conversion of the basic dyes Nile Blue and Brilliant Cresyl Blue, with Hansch pi values of -2.4 and -3.6, into their oxazone derivatives, with Hansch pi values of +4.4, and 3.6, generates lipid staining compounds. Also in keeping with this correlation of lipophilia with Hansch pi values greater than +1.0 is the occurrence of artifactual lipid staining in enzyme histochemistry. Such artifacts can arise for instance when using certain naphthyl substrates which give rise to naphthoic intermediate reaction products, or when tetrazolium salts as used as visualising agents, yielding formazans as final reaction products. The Hansch pi values of the naphthols and formazans generated histochemically typically fall into the lipophilic range. Another artifact of lipid staining is the staining of basophilic entities, such as cell nuclei, by fat stains which carry amino substituents. Calculation of the Hansch pi values for protonated (and hence cationic) derivatives of such dyes yields values typical of basic dyes.

Animals↗

Reduction of tetrazolium salt XTT by aminoreductone formed during the Maillard reaction of lactose.

Lactose, a reducing disaccharide abundant in milk, reacts extensively with the amino groups of protein through the Maillard reaction. The Maillard reaction products showed 3'-[1-[(phenylamino)carbonyl]-3,4-tetrazolium]bis(4-methoxy-6-nitro)benzensulfonic acid hydrate (XTT) reducibility. The objective of this study was to clarify the Maillard reaction products responsible for the XTT reducibility. When lactose and butylamine were heated at 100 degrees C, the characteristic UV maximum at 320 nm was recognized and the relationship between the XTT reducibility and the compound with absorption maximum at 320 nm was investigated. The time course and the dependence on the heating temperature of the formation of the compound with absorption maximum at 320 nm were similar to those of the XTT reducibility. Their relationship showed a significant correlation (r = 0.967, n = 19). Furthermore, the spectrum change of the heated model solution by the addition of XTT suggested that the compound with absorption maximum at 320 nm would be involved in the reduction of XTT. Because the compound with absorption maximum at 320 nm was identified as an aminoreductone, 1-(butylamino)-1,2-dehydro-1,4-dideoxy-3-hexulose, by NMR analysis, it can be concluded that this was the main XTT-reducing substance.

Animals↗

Comparison of the effects of molsidomine, nitroglycerin and isosorbide dinitrate on experimentally induced coronary artery thrombosis in the dog.

Platelet activation and aggregation in the coronary circulation may be important in the pathogenesis of myocardial ischemia. Molsidomine (M), isosorbide dinitrate (ISDN) and nitroglycerin (NTG) have been found to inhibit platelet aggregation in vitro. In the present study, the activity of these compounds was investigated in a model of coronary artery thrombosis in vivo. Dogs were anesthetized, thoracotomized, and their heart was exposed. An electrode was inserted into the left circumflex coronary artery and set to rest on the intima. Electrical stimulation (9 V, 150 microA) lasted for 6 h. Compounds (each in 2 dose levels) were given as an i.v. infusion starting 30 min after the beginning of the stimulation and lasting for the duration of the experiment. All control (saline-treated) animals underwent thrombotic occlusion of the coronary artery as assessed by flow measurement. On the other hand, 2/8 dogs treated with the lower M dose and 4/8 dogs treated with the higher M dose did not have a coronary occlusion. Neither ISDN nor NTG, at both doses, prevented the coronary occlusion. In control animals thrombus wet weight was 74.43 +/- 11.25 mg. M reduced the thrombus weight in a dose-related manner, while ISDN (marginally) and NTG (significantly at the higher dose) increased this parameter. Following the coronary thrombosis, all control animals developed myocardial infarcts as assessed by the tetrazolium technique. Similarly all animals treated with ISDN and with NTG (at both doses) showed infarcts. However, 3/8 M-dogs did not have a myocardial infarction in the lower as well as in the higher dose groups. The hemodynamic changes induced by the 3 compounds were similar in magnitude. Thus M but not ISDN or NTG showed in this in-vivo study antithrombotic and consequently antiischemic activity.

Animals↗

Evaluation of cytotoxicity of MTAD using the MTT-tetrazolium method.

Previous studies have shown that MTAD (a mixture of a tetracycline isomer, an acid, and a detergent) is an effective antibacterial irrigant as a final rinse to remove the smear layer from the instrumented surface of root canals. In this investigation we examined the cytotoxicity of MTAD compared with that of commonly used irrigants and medications. L929 fibroblasts were grown on cell culture plates and were placed in contact with various concentrations of test irrigants and medications. The cytotoxicity of these materials was evaluated 24 h after incubation using MTT assay. Means and standard deviations of absorbance were calculated for each group and statistically analyzed to determine presence or absence of significant difference between the means. The 50% inhibitory dose values were calculated, ranked, and statistically analyzed using the sign interval for median. Based on our results it seems that MTAD is less cytotoxic than eugenol, 3% H2O2, Ca(OH)2 paste, 5.25% NaOCl, Peridex, and EDTA and more cytotoxic than 2.63%, 1.31%, and 0.66% NaOCl.

Animals↗

Cytotoxic activity of methanol extracts from Basidiomycete mushrooms on murine cancer cell lines.

Crude methanol extracts of 58 mushroom species were screened for their cytotoxic activities against two murine cancer cell lines, L1210 and 3LL, using the tetrazolium assay. A majority of extracts (74%) exhibited IC50 > 100 microg/ml against both cell lines. A most marked activity against one of the cell lines was noted for nine species (14% of the tested species). While Amanitales and Russulales tested were not found active, Polyporales and Boletales gave better results. Four species exhibited a significant cytotoxic activity (IC50 < or = 20 microg/ml) against at least one of the two murine cancer cell lines (Ganoderma lucidum, Meripilus giganteus, Suillus granulatus, S. luteus). The last one had never been investigated for its cytotoxic compounds before.

Animals↗

Violacein and its beta-cyclodextrin complexes induce apoptosis and differentiation in HL60 cells.

Violacein, a pigment isolated from Chromobacterium violaceum, has been reported to have multiple biological activities including in vitro antitumor effects. Certain anticancer agents are known to induce apoptosis in human tumor cell lines. In this work, our aim was to investigate the effectiveness of violacein/beta-cyclodextrin (beta-CD)-containing systems to produce lethal effects in the human promyelocytic leukemia cell line HL60. Using the MTT tetrazolium reduction test, IC(50) for the inclusion complexes (1:1 and 1:2 violacein:beta-CD molar ratios) and violacein alone were less than 1 microM. Violacein and violacein/beta-CD complexes were able to induce NBT reduction. Moreover, by using the Feulgen reaction, all the compounds were found to trigger apoptosis in HL60 cells, inducing around 35% of DNA fragmentation, as analyzed through the diphenylamine assay. In addition, caspases seem to play an important role in the activation of the executioner phase of apoptosis induced by violacein and its derivatives.

Apoptosis↗