Role of Extracellular Signal-Regulated Kinase 1/2 and Reactive Oxygen Species in Toll-Like Receptor 2-Mediated Dual-Specificity Phosphatase 4 Expression / 영남의대학술지

BACKGROUND: Toll-like receptors (TLRs) are well-known pattern recognition receptors. Among the 13 TLRs, TLR2 is the most known receptor for immune response. It activates mitogen-activated protein kinases (MAPKs), which are counterbalanced by MAPK phosphatases [MKPs or dual-specificity phosphatases (DUSPs)]. However, the regulatory mechanism of DUSPs is still unclear. In this study, the effect of a TLR2 ligand (TLR2L, Pam3CSK4) on DUSP4 expression in Raw264.7 cells was demonstrated. METHODS: A Raw264.7 mouse macrophage cell line was cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% antibiotics (100 U/mL penicillin and 100 g/mL streptomycin) at 37degrees C in 5% CO2. TLR2L (Pam3CSK4)-mediated DUSP4 expressions were confirmed with RT-PCR and western blot analysis. In addition, the detection of reactive oxygen species (ROS) was measured with lucigenin assay. RESULTS: Pam3CSK4 induced the expression of DUSP1, 2, 4, 5 and 16. The DUSP4 expression was also increased by TLR4 and 9 agonists (lipopolysaccharide and CpG ODN, respectively). Pam3CSK4 also induced ERK1/2 phosphorylation and ROS production, and the Pam3CSK4-induced DUSP4 expression was decreased by ERK1/2 (U0126) and ROS (DPI) inhibitors. U0126 suppressed the ROS production by Pam3CSK4. CONCLUSION: Pam3CSK4-mediated DUSP4 expression is regulated by ERK1/2 and ROS. This finding suggests the physiological importance of DUSP4 in TLR2-mediated immune response.

Establishment of BCRP expressed pig kidney cell line LLC-PK1/BCRP and its biological profile / 药学学报

To establish a pig kidney cell line LLC-PK1/BCRP in which human breast cancer resistance protein was highly expressed, the expression vector pcDNA3.1(+)-BCRP which contained BCRP gene was constructed and transfected into LLC-PKI cells via liposomes. After selecting with G418, population doubling time, flow cytometry and Western blotting analysis were used to evaluate the cell line. MTT assays were employed to determine the drug resistance index of mitoxantrone and doxorubicin. Invert fluorescent microscope was used to observe the efflux of fluorescence dye Hoechst 33342 by BCRP, furthermore, the BCRP's inhibitor GF120918 was applied to reverse the efflux of Hoechst 33342. The experiment results showed that the expression of BCRP protein increased in LLC-PK1/BCRP cell. The population doubling time of LLC-PK1/BCRP cell was a little longer than that of the parental cell LLC-PK1. The resistance indexes to mitoxantrone and doxorubicin were 51.95 and 6.09 times, respectively, higher than LLC-PK1 cell. The efflux of Hoechst 33342 was significantly enhanced and could be reversed by GF120918. So a LLC-PK1/BCRP cell line was established, which highly expressed BCRP protein successfully. This cell line could be a valuable model to further investigate the biological profile of BCRP and select the substrate and inhibitor of BCRP.

Redox Regulating Protein APE1/Ref-1 Expression is Increased in Abdominal Aortic Coarctation-induced Hypertension Rats

BACKGROUND: Aim of study is designed to investigate whether apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE1/Ref-1) expression is changed in abdominal aortic coarctation models. METHODS: Male Sprague-Dawley rats were randomly assigned with abdominal aortic coarctation, repaired group, sham, and control groups. Endothelial function was assessed with endothelium-dependent relaxations. Detection of superoxide anion and lipid peroxidation was performed by lucigenin chemiluminescence and thiobarbituric acid-reactive substances assay. APE1/Ref-1 expression was measured with Western blot and immunohistochemistry. RESULTS: In anesthetized condition, the abdominal aortic coarctation rats showed hypertension as systolic/diastolic arterial pressure of 171/114 mm Hg, compared with 114/94 mm Hg of control. Endothelium-dependent relaxations were significantly impaired in the aortic coarctation which was recovered in 1 week after coarctation repair. Superoxide production and lipid peroxidation were elevated in aortic coarctation rats. In immunohistochemistry, APE1/Ref-1 expressions were increased at aorta and kidney in aortic coarctation rats. Increased APE1/Ref-1 expression in aorta was recovered by repair of coarctation. CONCLUSIONS: Taken together, it suggests that APE1/Ref-1 expression was increased in aortic coarctation-induced hypertensive rats, suggesting a biomarker for hypertension. Impaired endothelium dependent relaxation in the aortic coarctation can be modulated by repair of coarctation or the modulation of blood pressure.

The transport of gastrodin in Caco-2 cells and uptake in Bcap37 and Bcap37/MDR1 cells / 药学学报

Gastrodin (GAS) is the major bioactive component of the extracts from the rhizome of Gastrodia elata Blume. The aim of this study is to investigate the transport of GAS in Caco-2 cells and the interaction of P-glycoprotein and GAS. The apparent permeability coefficients (Papp) of GAS were measured as a function of directions and concentrations. It was demonstrated that the efflux ratio was < 2.0 over the range of 50-500 micromol x L(-1) of GAS from bi-directional transport studies. The transport rate of GAS was dependent on the concentrations. Papp of GAS was not affected by transport directions, GAS concentration or the classical inhibitors of P-glycoprotein (verapamil and GF 120918). The cellular accumulation of GAS in Bcap37/MDR1 cells transected with hMDR1 gene, was similar to that in Bcap37 cells. The accumulation in both cell lines was concentration dependent. GAS did not affect the accumulation of Rhodamine 123 in Bcap37/MDR1 cells over the range of 50-500 micromol x L(-1). It indicated that the transport of GAS in Caco-2 cell monolayers mainly is by passive paracellular transport pathway. P-glycoprotein did not participate in the absorption of GAS in the intestine or the transport across the blood-brain barrier.

Synthesis and antimicrobial activity of some new substituted 9-[1H-pyrazolo[3,4-b] quinolin-1-YL] acridines

2-Chloro-6-methyl/methoxy-3-formylquinoline upon condensation with substituted 9- hydrazino-acridine afforded corresponding hydrazones which were cyclized under alkaline condition to give 9-[6-substituted-1H-pyrazolo[3,4-b]quinolin-1-yl] acridine analoges. All of the synthesized compounds were characterized by their elemental analysis, IR and NMR spectral studies. Biological screening of the prepared compounds have been screened on some strain of bacteria and fungus

Synthesis and antineoplastic activity of certain triazene and triazeno-acridine combilexin derivatives

Four series of bifunctional ligands have been synthesized as DNA-binding combilexins. These novel agents contain a triazeno-benzene sulfonamide linker moiety that is attached to an intercalating acridine or acridone chromophore by a functionalized amide or ester residue. In order to obtain these combilexins three series of the anticipated antitumor triazeno-benzene sulfonamide were synthesized. The synthesis and bioscreening of the new antineoplastic compounds are depending on the structural correlation with several reported antineoplastic acridines. 2-Chlorobenzoic acid was reacted with anthranilic acid to give N-[2-carboxyphenyl] anthranilic acid which upon cyclodehydration with sulfuric acid afforded 9-oxo-9, 10-dihydroacridine-4-carboxylic acid, [acridone-4-carboxylic acid] 8. This latter intermediate has been converted to 9-chloroacridine carbonyl chloride 9 using thionyl chloride. Selective substitution of 9 with derivatives of 4-[piperazine-l-yldiazenyl] benzenesulfonamides 4a-e or derivatives of 4-[2-hydroxyethyl] piperazine-l-yl]diazenyi] benzenesulfonamides 5a-e to yield their 9-chloroacridine-4-carboxamides 10a-e and 9-chloroacridine-4-carboxylic acid esters 13a-e respectively. Those intermediates have been reacted either with different sulfonamides to give derivatives of 4-[4-[4-[4 sulfamoylphenyldiazenyl] piperazine-l-carbonyl]-9-ylamino] benz-enesulfonamides 11a-h and derivatives of 2-[[4-[4-sulfamoyl-phenyl]diazenyl]piperazine-l-yl]ethyl 9-[4-sulfamoylphenyl-amino]-9,10-dihydroacridine-4-carboxylates 14a-i respectively or subjected to mild acid hydrolysis to yield derivatives of 4-[4-[[9-oxo-9,1 Q-dihydroacridine-4-carbonyl]piperazine-l-yl]diazenyl]-benzenesulfonamide 12a-e and derivatives of 2-[4-[[4-sulfamoyl-phenyl]diazenyl]piperazine-l-yl]ethyl-9-oxo-9,10-dihydroacridine-4-carboxylate 15a-e respectively. Besides, the synthesis of derivatives of 4-[piperazine-l-yldiazenyl] benzenesulfonamides 4a-e and derivatives of 4-[2-hydroxyethyl]piperazine-1-yl] diazenyl] benzenesulfonamides 5a-e has been achieved via diazotization of various substituted benzene sulfonamides with sodium nitrite and hydrochloric acid followed by various amines coupling to yield the target triazeno-benzene sulfonamides. Fourteen new compounds were selected for screening their antitumor activity against breast cell line in National Cancer Institute. Six of them were found to be active as antitumor agents, while two were found to be mild active

New antimicrobial 9-[p-heterocyclo-substituted anilino]-tetrahydroacridines

The chemistry of tetrahydroacridines is of continuous interest, as they are associated with pharmacological activities[1-9]. Some members of this class of compounds are used as memory-enhancing agents for treating Alzheimer disease [1, 2] acetylcholine esterase inhibitors[3-5], DNA-binding agents[6], antimicrobial agents[7] and as amoebicides[7-9]. The present work deals with the synthesis of a new series of 9-[p-[4-aryl- 3-cyano-2-iminopyridin-6-yl] anilino]-1, 2, 3, 4-tetrahydroacridines [3] and their 2-oxo-[or thioxo]-pyridinylanilino derivatives 4 and 5, besides other related products 7-12 to be evaluated against bacteria and fungi. Synthesis was achieved by allowing 9-chloro-1, 2, 3, 4-tetrahydroacridine [1][10] to react with p-aminoacetophenone to give the 9-[p-acetylanilino] derivative 2. The one pot reaction of 2 with malononitrile, ammonium acetate and the appropriate aromatic aldehyde afforded the corresponding 9-[p-[4-aryl-3-cyano-2-iminopyridin-6-yl]-anilino]-1, 2, 3, 4-tetrahydroacridines [3a-e], respectively [Route a]. Similarly, reaction of 2 with ethyl cyanoacetate, ammonium acetate and the appropriate aromatic aldehyde in n-butanol afforded the corresponding 9-[p-[4-aryl-3-cyano-2[1H]-oxo-pyridin-6-yl] anilino]-1, 2, 3, 4-tetrahydroacridines [4a-d], respectively. On the other hand, the reaction of an ethanolic mixture of 2 with substituted arylmethylene cyanothioacetanilides [5][11, 12] in the presence of ammonium acetate gave the corresponding 3-cyano-4-arylpyridin-2[1H]-thiones [6a-d], respectively [Route a]. Also reaction of 1 with ethyl-p-aminobenzoate gave the corresponding ethyl-p-[1, 2, 3, 4-tetra-hydroacridin-9-yl] aminobenzoate [7] which upon reaction with hydrazine hydrate afforded the corresponding acid hydrazide 8. [Route b]. In addition, acetylation of 2 was accomplished by heating it with ethyl acetate in the presence of sodium metal to give p-[[1, 2, 3, 4-tetrahydroacridin-9-yl]amino]acetylacetophenone [9]. Bromination of 2 afforded 9-[p-bromo-acetyl anilino]-1, 2, 3, 4-tetrahydroacridine [10] which upon reaction with thiourea gave the corresponding 9-[p-[2-aminothiazol-4-yl]aniline-1, 2, 3, 4-tetrahydroacridine [11] was hydrobromide salt, while reaction of 10 with malononitrile afforded p-[1, 2, 3, 4-tetrahydroacridin-9-yl]amino-benzoylmethyl malononitrile [12] [Route c]

Lucigenin chemiluminescence assay as an adjunctive tool for assessment of various stages of inflammation: a study of quiescent inflammatory cells.

A simple, fast, precise and biologically relevant toxicity assay for screening cytotoxicity of minerals would have distinct advantages due to its cost benefits and relative savings in time. Furthermore, a bioassay to differentiate acute and chronic in vivo pulmonary reactions could have potential value as predictors of fibrogenicity and pathogenicity. In this study we examined the potential use of lucigenin as a probe to evaluate the correlation between chemiluminescence (CL) generated by alveolar macrophages with the known cytotoxicity and patho genicity by conventional bioassays. In this study, we used small doses of dust (20 microg) to minimize cellular overload and to maintain homeostasis. Crystalline silica a highly fibrogenic dust was used as positive control and results are compared with those for bentonite, kaolin and talc. Among the three minerals compared with silica, bentonite was more reactive (27%) in CL assay and declined sharply compared to other minerals. This sudden decline in bentonite CL is caused by cytotoxicity leading to cell death. CL-induced by talc was comparable to silica and declines slowly. Kaolin on the other hand produced relatively a weaker (25%) CL compared to silica. Our data using relatively low doses of dust suggest that the CL assay may have a better predictive value in cytotoxicity evaluations compared to conventional toxicity assays.

9-(4-ethoxycarbonylphenoxy)-6,7-dimethoxy-1,2,3,4-tetrahydro acridine inhibits free radical induced rat cortical neuron cytotoxicity and cerebral ischemia injury / 药学学报

<p><b>AIM</b>To study the effects of 9-(4-ethoxycarbonylphenoxy)-6,7-dimethoxy-1,2,3,4-tetrahydro acridine (EDT) on free radical induced injury in primary cultured rat cortical neuron and cerebral ischemia in mice.</p><p><b>METHODS</b>In primary rat cortical neuron, free radical injury model was established by 10 mumol.L-1 H2O2. The content of malondiadehyde (MDA) and activity of superoxide dismutase (SOD) in cells were investigated. Chronic cerebral ischemia model was produced by occlusion of one carotid artery and pneumogastric nerve in mice. The step down test was adopted to investigate the effect of EDT on the memory impairment. The cerebra morphology and MDA, NO content and SOD activity in mice cerebra were detected.</p><p><b>RESULTS</b>In primary rat cortical culture, 0.01-3 mumol.L-1 EDT concentration-dependently inhibited the formation of MDA and reduction of SOD activity induced by 10 mumol.L-1 H2O2. In chronic cerebral ischemia, EDT 2.5, 5 and 10 mg.kg-1 ig for 5 d greatly improved the memory impairment, reduced NO efflux and MDA content, while increased SOD activity in mice cerebra.</p><p><b>CONCLUSION</b>EDT was found to protect neurons from H2O2-induced neurotoxicity and inhibit chronic cerebral ischemia mediated injury and memory impairment in mice.</p>

Synthesis, molecular modeling and antitiumor activity of some substituted acridine derivatives

Nine series of new acridine derivatives of anticipated antineoplastic activity were synthesized. The first series consists of N,N'-1, 2-bis [9-[4-substituted sulfamoylphenyl] amino-acridine-4-carboxamide] ethylenediamine. The second comprises of N,N'-1,4-bis [9-[4- substituted sulfamoylphenyl] aminoacridine-4-carboxamide] phenylenediamine. The third and fourth are 1-[4-substituted sulfamoylphenyl] aminoacridine-4-carboxylic acid [4-substituted sulfamoylphenyl] amide. The fifth and sixth are 9-ox-1-[4-substituted amino sulfamoylphenyl]-9-10- dihydroacridine-4-carboxylic acid [4- substituted sulfamoylphenyl] amide. The seventh is 2-substituted phenyl-2,6-dihydropyrazolo [3,4,5-kl] acridine-5-carboxylic acid [4- substituted sulfamoylphenyl] amide. The eighth is 4-[5-[N'- phenylhydrazinocarbonyl-6H-pyrazolo [3,4,5-kl acridine-2-yl]-N- substituted benzene sulfonamide. The last one belongs to 2- substituted phenyl-2,6-dihydropyrazolo [3,4,5-kl] acridine-5-carboxyli acid [4-substituted sulfamoylphenyl] hydrazide

Synthesis and antitumor activity of some hydrazinoacridine hydrazinoacridonanil and 9-oxo-9, 10-dihydroacridin-4-carboxazide derivatives

Six series of acridine derivatives of anticipated antitumor activity have been designed and synthesized. The first comprised 9-[4 sulfamoylphenyl] hydrazinoacridine. The second consisted of 9-[4-sulfamoylphenyl] hydrazinoacridonanil. The third included N- substituted 9-[4 sulfamoylphenyl] hydrazinoacridine-4-yl [N-[4-sulfamoylphenyl]] carboxamide. While the fourth and the fifth included N substituted 9-[4-sulfamoylphenyl]] hydrazinoacridin-4-yl[N- sulfamoylphenyl]] carboxazide derivatives; and the sixth included 9-oxo-9, 10-dihydroacridin-4-yl [N-[4-sulfamoylphenyl]] carboxazide derivatives. The rationale behind the synthesis of these compounds, their methods of synthesis as well as the antitumor activities have been presented

Synthesis of some new 9-substituted acridines of possible Antimicrobial activity

A series of 9-substituted acridines has been synthesized utilizing 3-[9-acridinyl] propanoylhydrazine, 9-[4-aminophenyl] acridine [II] and 9-chloromethylacridine [III] as starting materials. The hydrazones Ic and Id as well as the Schiff bases IIa-IIc were produced via condensation of the acid hydrazide Ib or the amine II with different aldehydes. The acid hydrazide Ib was also reacted with some acid anhydride to afford the pyridazinone derivatives Ie and If. In addition, 9-chloromethylacridine [III] was reached with glycine, sulfanilamide or sulfadiazine to give the corresponding N-alkylated derivative IIIa-IIIc. The structure of the synthesized products was substantiated by spectroscopic data and elemental analyses. Some of the prepared compounds were found to possess antimicrobial activity

Synthesis and antitumor activity of some 9- [4-substituted sulfamoylphenylamino] acridine-4-hydroxyalkylcarboxamide derivatives: Part II1

A series of 9-[4-substituted sulfamoylphenyl-amino] acridine-4-hydyroxyalkyl carboxamide derivatives has been synthesized in order to study the SAR of this class of compounds. The prepared final compounds have been evaluated for antitumor activity in NIH, Bethesda, Maryland, USA. No significant activity has been observed

Antischistosomal activity of acridanone-hydrazones in Cebus monkeys experimentally infected with the SJ strain of Schistosoma mansoni

In this study, four compounds where utilized at the dose of 12.5mg/kg body weight, p.o., to treat Cebus monkeys experimentally infected with about 200 cercariae of Schistosoma mansoni (SJ strain), via transcutaneous route. The oograms performed with rectal snips, as well as stool examinations carried out periodically, showed no viable eggs of the parasite, from day 29 to 226 post-treatment. The perfusion undertaken after killing the animals showed absence of worms in the treated monkeys, whereas 83 worms were recovered from the control, thus corroborating the results obtained by means of oograms and coproscopy. These results confirm the efficacy of 9-acridanonehydrazones previously tested against the LE strain of S. mansoni. The low curative dose and apparent absence of toxicity render these drugs an important therapeutic reserve, taking into consideration the reports on the resistance of S. mansoni to the modern drugs oxamiziquine and praziquantel.

Activity of 9-acridanone-hydrazone drugs detected at the pre-postural phase, in the experimental schistosomiasis mansoni

The compound Ro-15.5458/000, derivative in the class of 9-acridanone-hydrazones, was found to be effective against Schistosoma mansoni in mice, killing almost all the skin schistosomules (24 hr after infection), when administered at the dose of 100 mg/kg. In experiments carried out with Cebus monkeys, the drug was shown to be fully effective at 25 mg/kg, 7 days after infection. These data, associated with the good results obtained earlier at the post-postural phase of schistosomiasis, allow the inference that this promising compound may be important in the set of antischistosomal drugs, depending on further toxicological and clinical tests.

Synthesis of some substituted 9-anilinoacridines and 4-anilinoquinolines of possible antitumor activity

3-amino-4-methoxybenzoie acid methyl ester and 3-amino- 4-methoxy-6- methane sulphonamidobenzoic acid methyl ester were condensed with 4, 7-dichloroquinoline, 4-chloroquinaldine, 9-chloroacridine and 2-methoxy-6,9-dichloro acridine. The anilide esters obtained were reduced to the corresponding alcohols from which the N-methylcarbamate derivatives were prepared as possible antitumour agents

A rapid diagnosis of malaria parasites by acridine orange staining method

The study was designed to find out the diagnostic value of Acridine orange staining method in detection of malaria parasite (Plasmodium falciparum) in comparison with the conventional thick and thin blood films. Thirty two P. falciparum and two P. vivax malaria cases were included in thestudy and the thick blood film and acridine orange staining methods were found to be more sensitive in detecting asexual and sexual parasites (gametocytes) than the thin film. It needs 2-4 weeks time to train a technician to beable to detect the parasites in thick and thin film methods, but acridine orange staining method can be transferred within an hour. Another advantage of acridine orange staining method is the shortest examination time, lasting only 45 seconds whereas the thick and thin blood films need 5 to 10 minutes. Hence, it is concluded that the acridine orange staining method is useful for quick diagnosis of malaria although accessary eauipment is required.

Synthesis and in vitro cytotoxic activity of certain new 9-acridinyl and 9-acridinylmethyl derivatives of amino acids

The synthesis of two series of 9-[N-substituted amino] acridines 1a and 9-[N[-substituted] aminomethyl] acridines 1b and their corresponding N-methyl quaternary salts 2a and 2b and N10 oxides 3a and 3b was reported. Preliminary testing for the in vitro cytotoxic activity of 30 representative examples against Ehrlich ascites carcinoma was carried out. The most active derivatives are those of arginine and glutamine. The N10-oxido derivatives have superior activity