Triterpenoids and an alkamide from Ganoderma tsugae.

Ganoderma tsugae is a medicinal mushroom. In a continual study on the bioactive constituents of this fungus, a new lanostanoid, 3ß-acetoxy-16α-hydroxy-24ξ-methyl-5α-lanosta-8,25-dien-21-oic acid, named tsugaric acid F (1) and a novel palmitamide, N-(3'α,4'ß-dihydroxy-2'ß-(hydroxymethyl)-1'ß-(cyclobutyl)palmitamide (2) were isolated and characterized from the fruit bodies of G. tsugae, and three novel seco-lanostanoids, 3,4-seco-8α,9α-epoxy-5α-lanosta-21-oic acid 3,4 lactone (5), 3,4-seco-5ß-lanosta-7,9(11),4(29)-trien-3,21-dioic acid-3-methyl ester (6), 3,4-seco-5ß-lanosta-7,9(11),4(29)-trien-3,21-dioic acid (7), and a known compound, 3-oxo-5α-lanosta-8-en-21-oic acid (4) were prepared from 3. The structures of new compounds, 1, 2, 5-7 were determined by spectroscopic methods. Compounds 1 and 4 showed inhibitory effects on xanthine oxidase (XO) with an IC50 values of 313.3 ± 80.0 and 43.9 ± 29.9 µM, respectively when 7 exhibited potent inhibitory effect on superoxide anion generation in rat neutrophils stimulated with formyl-Met-Leu-Phe (fMLP)/cytochalasin B (CB) with an IC50 values of 1.3 ± 0.2 µM. Compounds 4-7 showed weak cytotoxic activities against PC3 cells. These results indicated that 4 and 7 may be used as cancer chemopreventive agents.

Inhibition of formyl peptide-stimulated phospholipase D activation by Fal-002-2 via blockade of the Arf6, RhoA and protein kinase C signaling pathways in rat neutrophils.

Three recently developed selective phospholipase D (PLD) inhibitors N-(2-(4-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidin-1-yl)ethyl)-2-naphthamide (VU0155056), (S)-N-(1-(4-(5-chloro-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidin-1-yl)propan-2-yl)-2-naphthamide (VU0155069), and N-(2-(4-oxo-1-phenyl-1,3,8-triazaspiro[4,5]decan-8-yl)ethyl)quinoline-3-carboxamide (VU0285655-1) inhibited O2 (•-) generation in formyl-Met-Leu-Phe (fMLP)-stimulated rat neutrophils. A novel 2-phenyl-4-quinolone compound 6-chloro-2-(2-chlorophenyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (Fal-002-2), which inhibited O2 (•-) generation, also reduced the fMLP- but not phorbol ester-stimulated PLD activity (IC50 16.0 ± 5.0 µM). Fal-002-2 attenuated the interaction of PLD1 with ADP-ribosylation factor (Arf) 6, Ras homology (Rho) A and protein kinase C (PKC) isoforms (α, ßI, and ßII), and also inhibited the membrane recruitment of Arf6 and RhoA in fMLP-stimulated neutrophils, but not in GTPγS-stimulated cell-free system. The cellular levels of GTP-bound Arf6 and GTP-bound RhoA were reduced by Fal-002-2. Fal-002-2 also attenuated the membrane recruitment of Rho-associated protein kinase 1, phosphorylation of myosin light chain 2 at Thr18/Ser19 and PLD1 at Thr147, and the interaction of Arf6 with both arfaptin 1 and phosphatidylinositol 4-phosphate 5-kinase 1A. The association between RhoA and Vav, the interaction of Vav with both Lyn and Lck, the membrane recruitment of Vav, and the phosphorylation of Vav at Tyr174, but not Src family at Tyr416, were all attenuated by Fal-002-2 in fMLP-stimulated neutrophils. These results indicate that Fal-002-2 is not a direct PLD inhibitor, but the inhibition of fMLP-stimulated PLD activity by Fal-002-2, which partly accounts for its suppression of O2 (•-) generation, is attributable to the blockade of both Arf6 and RhoA activation and attenuation of the interaction of Arf6, RhoA and PKC isoforms with PLD1 in rat neutrophils.

Inhibition of formyl peptide-stimulated superoxide anion generation by Fal-002-2 occurs mainly through the blockade of the p21-activated kinase and protein kinase C signaling pathways in ratneutrophils.

In formyl-Met-Leu-Phe (fMLP)-stimulated rat neutrophils, a synthetic compound, 6-chloro-2-(2-chlorophenyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (Fal-002-2), inhibited superoxide anion (O2(•-)) generation with an IC50 value of about 11µM, which was not mediated by scavenging the generated O2(•-) or by a cytotoxic effect on neutrophils. Fal-002-2 effectively attenuated the phosphorylation of Ser residues in p47(phox) and the association between p47(phox) and p22(phox) in fMLP-stimulated neutrophils. The interaction of p47(phox) with protein kinase C (PKC) isoforms (α, ßI, ßII, δ and ζ) was attenuated by Fal-002-2 with a similar IC50 value to that required for inhibition of O2(•-) generation, whereas Fal-002-2 had no prominent effect on PKC isoform membrane translocation and did not affect the kinase activity. Moreover, Fal-002-2 had no effect on the phosphorylation of Akt and downstream glycogen synthase kinase-3ß, only slightly affected the intracellular free Ca(2+) concentration, phosphorylation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase (MAPK), but effectively attenuated the downstream MAPK-activated protein kinase-2 phosphorylation. The interaction of p21-activated kinase (PAK) 1with p47(phox), phosphorylation of PAK1 (Thr423/Ser144) and the membrane recruitment of PAK1 were effectively inhibited by Fal-002-2. Fal-002-2 also blocked the activation of Rac1 and Cdc42 in a concentration range that effectively inhibited PAK activation. Taken together, these results suggest that Fal-002-2 inhibits fMLP-stimulated O2(•-) generation in neutrophils mainly through the blockade of PKC and PAK signaling pathways and partly through p38 MAPK signaling.

p38 Mitogen-activated protein kinase and extracellular signal-regulated kinase signaling pathways are not essential regulators of formyl peptide-stimulated p47(phox) activation in neutrophils.

Three structurally unrelated p38 mitogen-activated protein kinase (MAPK) inhibitors, (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole (SB203580), 1-5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-(2-morpholin-4-yl-ethoxy)naphthalen-1-yl] urea (BIRB 796) and 5-(2,6-dichlorophenyl)-2-[2,4-difluorophenyl]thio]-6H-pyrimido[1,6-b]pyridazin-6-one (VX 745) showed approximately 40% inhibition of formyl-Met-Leu-Phe (fMLP)-stimulated neutrophil superoxide anion (O2(•-)) generation at concentrations that greatly diminished p38 MAPK activity. However, a significant inhibition of p47(phox) activation occurred at concentrations much higher than the corresponding IC50 values of these inhibitors in blocking p38 MAPK activity. 4-Ethyl-2(p-methoxyphenyl)-5-(4'-pyridyl)-IH-imidazole (SB202474), an inactive analogue of SB203580, at a concentration (30µM) which significantly attenuated p38 MAPK activity, had no effect on p47(phox) activation, whereas it inhibited O2(•-) generation with an IC50 value of approximately 16µM. Moreover, both SB203580 and BIRB 796 had no effect on protein kinase B (PKB)/Akt Ser473 phosphorylation and S100A9 protein membrane translocation at concentrations that effectively blocked p38 MAPK activity. Pretreatment of cells with two structurally unrelated MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors, 2-(2-amino-3-methoxy-phenyl)-chromen-4-one (PD 98059) and 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126), at concentrations that effectively blocked MEK activity, attenuated p47(phox) phosphorylation but did not affect the recruitment of p47(phox) to p22(phox) or O2(•-) generation. Both p47(phox) activation and O2(•-) generation were attenuated by a protein kinase C (PKC) inhibitor 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF 109203X) in the concentration range that effectively blocked PKC activity. Taken together, these results suggest that the ERK-mediated Ser phosphorylation of p47(phox) is not implicated in the assembly of NADPH oxidase or O2(•-) generation, and that O2(•-) generation is partly attributable to p38 MAPK signaling through mechanisms other than p47(phox) activation, Akt activation and S100A9 membrane recruitment in fMLP-stimulated neutrophils.

The signaling mechanisms mediating the inhibitory effect of TCH-1116 on formyl peptide-stimulated superoxide anion generation in neutrophils.

In fMLP (formyl-Met-Leu-Phe)-stimulated rat neutrophils, a mixture of regioisomers benzo[a]furo[2,3-c]phenazine-10-carboxylic acid and benzo[a]furo[2,3-c]phenazine-11-carboxylic acid (TCH-1116) inhibited O(2)(-) (superoxide anion) generation, which was not mediated by scavenging the generated O(2)(-) or by a cytotoxic effect on neutrophils. TCH-1116 had no effect on the arachidonic acid-induced NADPH oxidase activation in a cell-free system, whereas it effectively attenuated the phosphorylation of Ser residues in p47(phox) and the association between p47(phox) and p22(phox) in fMLP-stimulated neutrophils. The interaction of p47(phox) with PKC (protein kinase C) isoforms (α, ßI, ßII, δ and ζ) was attenuated by TCH-1116, whereas TCH-1116 did not affect the PKC isoforms membrane translocation, phosphorylation (Ser660) and kinase activity. TCH-1116 effectively attenuated the association between PKB/Akt (protein kinase B) and p47(phox), Akt phosphorylation (Thr308/Ser473) and kinase activities of Akt and human recombinant PDK (3-phosphoinositide-dependent kinase) 1, whereas it had no effect on recruitment of Akt, phospho-PDK1 (Ser241) and p110γ to membrane. Moreover, the interaction of p21-activated kinase (PAK) 1 with p47(phox) and the phosphorylation of PAK1 (Thr423 but not Ser144) were inhibited by TCH-1116, but without affecting the membrane recruitment of PAK1. The cellular cyclic AMP level was not changed by TCH-1116. Taken together, these results suggest that TCH-1116 inhibits fMLP-stimulated O(2)(-) generation in rat neutrophils through the blockade of PKC, Akt and PAK signaling pathways.

Identification of furo[3', 2':3,4]naphtho[1,2-d]imidazole derivatives as orally active and selective inhibitors of microsomal prostaglandin E(2) synthase-1 (mPGES-1).

This study describes the synthesis and anti-inflammatory effects of furo[3', 2':3,4]naphtho[1,2-d] imidazole derivatives. Among these furo[3', 2':3,4]naphtho[1,2-d]imidazole derivatives, 2-(4-methoxyphenyl)furo [3', 2':3,4]naphtho[1,2-d]imidazole (12) exhibited a strong inhibitory activity against LPS-induced PGE(2) production, with an IC(50) value of 47 nM. Compound 12 is then further examined for its inhibitory effects in the protein expression of COX-2 and microsomal prostaglandin E(2) synthase-1 (mPGES-1) in Raw 264.7 cells. Our results indicate that compound 12 was capable against inhibiting LPS-induced mPGES-1 protein expression at a concentration of 1.0 µM and no inhibitory effect in COX-2 expression. The sepsis-induced PGE(2) production in rat serum decreased ~250% by the pretreatment of 12 at 10 mg/kg. These results are especially important since compound 12 exhibited good oral bioavailability (72%) and was not cytotoxic at a concentration of 10.0 µM. Therefore, compound 12 is a highly selective mPGES-1 inhibitor that can serve as a lead for the development of novel oral anti-inflammatory drug candidates.

Signaling mechanisms of inhibition of phospholipase D activation by CHS-111 in formyl peptide-stimulated neutrophils.

A selective phospholipase D (PLD) inhibitor 5-fluoro-2-indolyl des-chlorohalopemide (FIPI) inhibited the O(2)(-) generation and cell migration but not degranulation in formyl-Met-Leu-Phe (fMLP)-stimulated rat neutrophils. A novel benzyl indazole compound 2-benzyl-3-(4-hydroxymethylphenyl)indazole (CHS-111), which inhibited O(2)(-) generation and cell migration, also reduced the fMLP- but not phorbol ester-stimulated PLD activity (IC(50) 3.9±1.2µM). CHS-111 inhibited the interaction of PLD1 with ADP-ribosylation factor (Arf) 6 and Ras homology (Rho) A, and reduced the membrane recruitment of RhoA in fMLP-stimulated cells but not in GTPγS-stimulated cell-free system. CHS-111 reduced the cellular levels of GTP-bound RhoA, membrane recruitment of Rho-associated protein kinase 1 and the downstream myosin light chain 2 phosphorylation, and attenuated the interaction between phosphatidylinositol 4-phosphate 5-kinase (PIP5K) and Arf6, whereas it only slightly inhibited the guanine nucleotide exchange activity of human Dbs (DH/PH) protein and did not affect the arfaptin binding to Arf6. CHS-111 inhibited the interaction of RhoA with Vav, the membrane association and the phosphorylation of Vav. CHS-111 had no effect on the phosphorylation of Src family kinases (SFK) but attenuated the interaction of Vav with Lck, Hck, Fgr and Lyn. CHS-111 also inhibited the interaction of PLD1 with protein kinase C (PKC) α, ßI and ßII isoenzymes, and the phosphorylation of PLD1. These results indicate that inhibition of fMLP-stimulated PLD activity by CHS-111 is attributable to the blockade of RhoA activation via the interference with SFK-mediated Vav activation, attenuation of the interaction of Arf6 with PLD1 and PIP5K, and the activation of Ca(2+)-dependent PKC in rat neutrophils.

New diterpenoids and cytotoxic and anti-inflammatory diterpenoids from Amentotaxus formosana.

Two new abietane diterpenoids, ramentoxide (1) and ramentoxidone (2) and a new icetexane diterpenoid, amentonone (3) were isolated from the barks of Amentotaxus formosana. The structures of 1-3 were determined by spectroscopic methods. Known compounds brevitaxin (4), and (+)-ferruginol (5) and ent-kaur-16-en-15-one (6) isolated from this plant revealed potent cytotoxic activity against human breast adenocarcinoma cells, MCF-7 cells with an IC(50) value of 0.08 ± 0.05 µg/mL, and significant anti-inflammatory activities, respectively.

Discovery of 3-(4-bromophenyl)-6-nitrobenzo[1.3.2]dithiazolium ylide 1,1-dioxide as a novel dual cyclooxygenase/5-lipoxygenase inhibitor that also inhibits tumor necrosis factor-alpha production.

In the present study we have discovered compound 1, a benzo[1.3.2]dithiazolium ylide-based compound, as a new prototype dual inhibitor of cyclooxygenase (COX) and 5-lipoxygenase (5-LOX). Compound 1 was initially discovered as a COX-2 inhibitor, resulting indirectly from the COX-2 structure-based virtual screening that identified compound 2 as a virtual hit. Compounds 1 and 2 inhibited COX-1 and COX-2 in mouse macrophages with IC(50) in the range of 1.5-18.1microM. Both compounds 1 and 2 were also found to be potent inhibitors of human 5-LOX (IC(50)=1.22 and 0.47microM, respectively). Interestingly, compound 1 also had an inhibitory effect on tumor necrosis factor-alpha (TNF-alpha) production (IC(50)=0.44microM), which was not observed with compound 2. Docking studies suggested the (S)-enantiomer of 1 as the biologically active isomer that binds to COX-2. Being a cytokine-suppressive dual COX/5-LOX inhibitor, compound 1 may represent a useful lead structure for the development of advantageous new anti-inflammatory agents.

Acetogenin and prenylated flavonoids from Helminthostachys zeylanica with inhibitory activity on superoxide generation and elastase release by neutrophils.

One new acetogenin, 6-hydroxy-8-pentadecyloxocane-2,7-dione ( 1), and four new prenylated flavonoids, 4''a,5'',6'',7'',8'',8''a-hexahydro-5,3',4'-trihydroxy-5'',5'',8''a-trimethyl-4 H-chromeno[2'',3'':7,6]flavone ( 2), 4''a,5'',6'',7'',8'',8''a-hexahydro-5,3',4',-trihydroxy-5'',5'',8''a-trimethyl-4 H-chromeno[2'',3'':7,8]flavone ( 3), 2-(3,4-dihydroxyphenyl)-6-((2,2-dimethyl-6-methylenecyclohexyl)methyl)-5,7-dihydroxy-chroman-4-one ( 4), and 2-(3,4-dihydroxy-2-[(2,6,6-trimethylcyclohex-2-enyl)methyl]phenyl)-3,5,7-trihydroxy-4 H-chromen-4-one ( 5), together with six known compounds, were isolated and purified from the rhizomes of Helminthostachys zeylanica by column chromatography and high performance liquid chromatography (HPLC) via bioactivity-guided fractionation isolation. The structures of the new isolates were elucidated by spectroscopic methods. Compounds 1, 3, and 5 showed inhibitory activities on either superoxide anion generation or elastase release by human neutrophils in response to formyl-L-methionyl-L-leucyl-L-phenylalanine/cytochalasin B (FMLP/CB).

Phloroglucinols inhibit chemical mediators and xanthine oxidase, and protect cisplatin-induced cell death by reducing reactive oxygen species in normal human urothelial and bladder cancer cells.

Phloroglucinols, garcinielliptones HA-HE (1-5), and C (6) were studied in vitro for their inhibitory effects on chemical mediators released from mast cells, neutrophils, and macrophages. Compound 6 revealed significant inhibitory effect on release of lysozyme from rat neutrophils stimulated with formyl-Met-Leu-Phe (fMLP)/cytochalasin B (CB). Compounds 3, 4, and 6 showed significant inhibitory effects on superoxide anion generation in rat neutrophils stimulated with (fMLP)/(CB), while compounds 1 and 5 revealed inhibitory effects on tumor necrosis factor-alpha (TNF-alpha) formation in macrophages stimulated with lipopolysaccharide (LPS). Compounds 1 and 3-6 showed inhibitory effects on xanthine oxidase (XO) and could inhibit the DNA breakage caused by O2(-*). Treatment of NTUB1 with 2 to 60 microM compound 3 and 5 microM cisplatin and SV-HUC1 with 9 to 60 microM 3 and 5 microM cisplatin, respectively, resulted in an increase of viability of cells. These results indicated that compounds 1 and 3-6 showed anti-inflammatory effects and antioxidant activities. Compound 3 mediates through the suppression of XO activity and reduction of reactive oxygen species (ROS), and protection of subsequent cell death.

Furo[3',2':3,4]naphtho[1,2-d]imidazole derivatives as potential inhibitors of inflammatory factors in sepsis.

Synthesis and anti-inflammatory effects of certain furo[3',2':3,4]naphtho[1,2-d]imidazole derivatives 12-18 were studied. These compounds were synthesized from naphtho[1,2-b]furan-4,5-dione (10) which in turn was prepared from the known 2-hydroxy-1,4-naphthoquinone (7) in a one pot reaction. Furo[3',2':3,4]naphtho[1,2-d]imidazole (12) was inactive (IC(50) value of >30 microM) while its 5-phenyl derivative 13, with an IC(50) value of 16.3 and 11.4 microM against lysozyme and beta-glucuronidase release, respectively, was comparable to the positive trifluoperazine. The same potency was observed for 5-furan derivative 16 with an IC(50) value of 19.5 and 11.3 microM against lysozyme and beta-glucuronidase release, respectively. An electron-withdrawing NO(2) substituted on 5-phenyl or 5-furanyl group led to the devoid of activity as in the cases of 14 and 17. Among them, compound 15 exhibited significant inhibitory effects, with an IC(50) value of 7.4 and 5.0 microM against lysozyme and beta-glucuronidase release, respectively. For the LPS-induced NO production, the phenyl derivatives 12-15 were inactive while the nitrofuran counterparts 17 and 18 suppress LPS-induced NO production significantly, with an IC(50) value of 1.5 and 1.3 microM, respectively, which are more active than that of the positive 1400 W. Compounds 16-18 were capable of inhibiting LPS-induced iNOS protein expression at a dose-dependent manner in which compound 18, with an IC(50) of 0.52 microM in the inhibition of iNOS expression, is approximately fivefold more potent than that of the positive 1400 W. In the CLP rat animal model, compound 18 was found to be more active than the positive hydrocortisone in the inhibition of the iNOS mRNA expression in rat lung tissue. The sepsis-induced PGE2 production in rat serum decreased 150% by the pretreatment of 18 in a dose of 10 mg/kg.

Inhibition of superoxide anion generation by CHS-111 via blockade of the p21-activated kinase, protein kinase B/Akt and protein kinase C signaling pathways in rat neutrophils.

In formyl-Met-Leu-Phe (fMLP)-stimulated rat neutrophils, 2-benzyl-3-(4-hydroxymethylphenyl)indazole (CHS-111) inhibited superoxide anion (O(2)(-)) generation, which was not mediated by scavenging the generated O(2)(-) or by a cytotoxic effect, and attenuated migration. CHS-111 had no effect on the arachidonic acid-induced NADPH oxidase activation or the GTPgammaS-stimulated Rac2 membrane translocation in cell-free systems, whereas it effectively attenuated the membrane recruitment of p40(phox), p47(phox) and p67(phox), phosphorylation of Ser residues in p47(phox), association between p47(phox) and p22(phox), and Rac activation in fMLP-stimulated neutrophils. Moreover, the phosphorylation and membrane recruitment of p21-activated kinase (PAK), PAK kinase activity and the interaction of PAK with p47(phox) were inhibited by CHS-111. CHS-111 effectively reduced Akt kinase activity and the association between Akt and p47(phox), moderately inhibited the membrane recruitment of Akt and phospho-PDK1, and slightly attenuated Akt (Thr308) phosphorylation, whereas it had no effect on Akt (Ser473) phosphorylation or p110gamma membrane translocation. The membrane recruitment of protein kinase C (PKC)-alpha, -betaI, -betaII, -delta and -zeta, PKC phosphorylation and PKC kinase activity was attenuated by CHS-111, whereas CHS-111 did not affect the phosphorylation of p38 mitogen-activated protein kinase (MAPK) or downstream MAPK-activated protein kinase-2. Higher concentrations of CHS-111 were required to decrease fMLP-stimulated intracellular free Ca(2+) concentration ([Ca(2+)](i)) elevation in the presence but not in the absence of extracellular Ca(2+), and to reduce cellular cyclic AMP but slightly increase cyclic GMP levels. Taken together, these results suggest that CHS-111 inhibits fMLP-stimulated O(2)(-) generation in rat neutrophils through the blockade of PAK, Akt and PKC signaling pathways.

Synthesis, anti-inflammatory, and antioxidant activities of 18beta-glycyrrhetinic acid derivatives as chemical mediators and xanthine oxidase inhibitors.

Twenty 18beta-glycyrrhetic acid (18beta-GA) derivatives 2-21 including 13 new 18beta-GA derivatives were synthesized and evaluated as anti-inflammatory and antioxidant agents. Compounds 7 and 20 with a 3,4-seco-structure and compound 6 with a lactone moiety showed potent inhibitory effect on superoxide anion generation in rat neutrophils response to fMLP/CB and PMA, respectively. Compound 6 with a lactone moiety revealed stronger inhibitory effect on XO activity than those of compounds 13 and 14 with a 3,4-seco-structure. Compound 14, a 30-isoproylcarbamoyl seco-compound exhibited potent inhibitory effect on NO accumulation and iNOS protein expression while compounds 3, 10, 13, 15, 17, and 21 revealed potent inhibitory effect on tumor necrosis factor-alpha (TNF-alpha) formation in RAW 264.7 cells in response to lipopolysaccharide (LPS). The cleavage of ring A of 3 attenuated the inhibitory effect on TNF-alpha formation in RAW 264.7 cells in response to LPS except for 17. The present results suggested these compounds were potential to be served as anti-inflammatory and antioxidant agents.

The influence of acetylshikonin, a natural naphthoquinone, on the production of leukotriene B4 and thromboxane A2 in rat neutrophils.

Both A23187 and formyl-Met-Leu-Phe (fMLP) induced the release of arachidonic acid and the production of thromboxane B(2) and leukotriene B(4) from rat neutrophils that were inhibited by acetylshikonin in a concentration-dependent manner. Acetylshikonin blocked exogenous arachidonic acid-induced leukotriene B(4) and thromboxane B(2) production in neutrophils and inhibited the enzymatic activity of ram seminal vesicles cyclooxygenase and human recombinant 5-lipoxygenase, whereas it had no effect on cytosolic phospholipase A(2) activity, in cell-free systems. 3-Morpholinosydnonimine- and 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13-HpODE)-mediated dihydrorhodamine 123 oxidation (to assess the lipid peroxide and peroxynitrite scavenging activity) was reduced by acetylshikonin. The membrane recruitment of cytosolic phospholipase A(2) was inhibited, but the phosphorylation of cytosolic phospholipase A(2) was enhanced, by acetylshikonin in the A23187-induced response. Acetylshikonin alone stimulated extracellular signal regulated kinase (ERK) phosphorylation and enhanced this response in cells stimulated with A23187 and fMLP. The phosphorylation of ERKs and cytosolic phospholipase A(2) was attenuated by U0126, a mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor. Acetylshikonin facilitated both A23187- and fMLP-mediated translocation of 5-lipoxygenase to the membrane. Acetylshikonin attenuated both fMLP- and ionomycin-mediated [Ca(2+)](i) elevation. These results indicate that the inhibition of eicosanoid production by acetylshikonin is due to the attenuation of cytosolic phospholipase A(2) membrane recruitment via the decrease in [Ca(2+)](i) and to the blockade of cyclooxygenase and 5-lipoxygenase activity.

Blockade of cytosolic phospholipase A(2) and 5-lipoxygenase activation in neutrophils by a natural isoflavanquinone abruquinone A.

Abruquinone A, a natural isoflavanquinone, suppressed A23187- and formyl-Met-Leu-Phe (fMLP)-induced production of thromboxane B(2) and leukotriene B(4) from rat neutrophils. This compound failed to inhibit the enzymatic activity of ram seminal vesicles cyclooxygenase (COX) and human recombinant 5-lipoxygenase (5-LO) in cell-free systems. Abruquinone A diminished the arachidonic acid release from [(3)H]arachidonic acid-loaded neutrophils stimulated with either fMLP or A23187, whereas it had no inhibitory effect on the cytosolic phospholipase A(2) (cPLA(2)) activity of neutrophil cytosolic fraction. Based on the Western blot analysis, the nuclear membrane recruitment of cPLA(2) and 5-LO was inhibited by abruquinone A in A23187- as well as in fMLP-stimulated cells. Moreover, the phosphorylation of both cPLA(2) and extracellular signal regulated kinases (ERKs) induced by fMLP and A23187 was attenuated by abruquinone A in a parallel concentration-dependent manner. Abruquinone A attenuated both fMLP- and ionomycin-mediated [Ca(2+)](i) elevation in a concentration range that inhibited the recruitment of cPLA(2) to nuclear membrane. These results indicate that the blockade of leukotriene B(4) production by abruquinone A implicates the attenuation of 5-LO membrane translocation. Inhibition of thromboxane B(2) production by abruquinone A is due to the attenuation of cPLA(2) membrane recruitment and/or cPLA(2) phosphorylation through the blockade of [Ca(2+)](i) elevation and ERK activation, respectively.

Antiplatelet effect and selective binding to cyclooxygenase by molecular docking analysis of 3-alkylaminopropoxy-9,10-anthraquinone derivatives.

In an effort to develop potent cytotoxic inhibitors of cyclooxygenase (COX), a series of cytotoxic 3-alkylaminopropoxy-9,10-anthraquinone derivatives was screened to evaluate their antiplatelet effect on washed rabbit platelets and human platelet-rich plasma (PRP). Thrombin, arachidonic acid (AA), collagen, and platelet-activating factor (PAF) induced platelet aggregations were potently inhibited by compounds 1, 2, and 3 (each at 300 microM). Of the compounds tested in human PRP, compounds 1, 8, and 10 showed significant inhibition of primary and secondary aggregation induced by epinephrine and had a weak inhibitory effect on cyclooxygenase-1 (COX-1). Molecular docking studies revealed that compounds, 1, 8, and 10 were bound in the active sites of COX-1. This indicated that the antiplatelet effect of these three compounds was partially mediated through the suppression of COX-1 activity and reduced thromboxane formation. It is concluded that the cytotoxic compounds 1, 8, and 10 may interfere the conversion of arachidonic acid to prostaglandin (PG)H(2) in the active site of COX-1.

Synthesis and cytotoxic, anti-inflammatory, and anti-oxidant activities of 2',5'-dialkoxylchalcones as cancer chemopreventive agents.

In an effort to develop novel anti-tumor, or cancer chemopreventive agents, a series of 2',5'-dialkoxylchalcones were prepared by Claisen-Schmidt condensation of appropriate acetophenones with suitable aromatic aldehyde. In vitro screening revealed low micromolar activity (IC(50)) against several human cancer cell lines. Selective compound 10 induced an accumulation of A549 cells in the G(2)/M phase arrest which was well correlated with inhibitory activity against tubulin polymerization. Cytotoxic compounds 3 and 12 showed significant inhibitory effects on NO production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophage-like cells while cytotoxic compound 10 revealed potent inhibitory effect on TNF-alpha formation in RAW 264.7 cells in response to LPS. Compounds 3 and 10 also showed significant inhibitory effects on xanthine oxidase. The present results suggested that compounds 3 and 10 were potential to be served as cancer chemopreventive agents.

Inhibition of nitric oxide production by the carbazole compound LCY-2-CHO via blockade of activator protein-1 and CCAAT/enhancer-binding protein activation in microglia.

Excessive nitric oxide (NO) production by activated microglia plays a critical role in neurodegenerative disorders. In this study, we found that 9-(2-chlorobenyl)-9H-carbazole-3-carbaldehyde (LCY-2-CHO) suppressed the NO production in lipopolysaccharide (LPS)/interferon-gamma (IFNgamma)-stimulated murine microglial N9 and BV-2 cells and in LPS-stimulated N9 cells and rat primary microglia. LCY-2-CHO had no cytotoxic effect on microglia. In activated N9 cells, LCY-2-CHO abolished the expression of inducible nitric oxide synthase (iNOS) protein and mRNA but failed to alter the stability of expressed iNOS mRNA and the enzymatic activity of expressed iNOS protein. LCY-2-CHO did not block DNA-binding activity of nuclear factor-kappaB (NF-kappaB) or cyclic AMP response element-binding protein (CREB), but abolished that of activator protein-1 (AP-1), CCAAT/enhancer-binding protein (C/EBP) and nuclear factor IL6 (NF-IL6). LCY-2-CHO attenuated the nuclear levels of c-Jun and C/EBPbeta, but not those of p65, p50, C/EBPdelta, signal transducer and activator of transcription-1 (STAT-1) or the nuclear expression of IFN regulatory factor-1 (IRF-1). LCY-2-CHO had no effect on the phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), MAPK-activated protein kinase-2 (MAPKAPK-2), STAT-1, CREB or c-Jun in LPS/IFNgamma-stimulated N9 cells, whereas it attenuated the phosphorylation of C/EBPbeta at Ser105 and Thr235 residues, which occurred concomitantly with LCY-2-CHO inhibition of C/EBPbeta expression and phosphorylation. Taken together, these results suggest that LCY-2-CHO inhibits NO production in microglia through the blockade of AP-1 and C/EBP activation.

Antiinflammatory triterpenoids and steroids from Ganoderma lucidum and G. tsugae.

The antiinflammatory properties of triterpenoids and steroids from both Ganoderma lucidum and Ganoderma tsugae were studied. Twelve compounds, including ergosta-7,22-dien-3beta-ol (1), ergosta-7,22-dien-3beta-yl palmitate (2), ergosta-7,22-dien-3-one (3), ergosta-7,22-dien-2beta,3alpha,9alpha-triol (4), 5alpha,8alpha-epidioxyergosta-6,22-dien-3beta-ol (5), ganoderal A (6), ganoderal B (7), ganoderic aldehyde A (8), tsugaric acid A (9), 3-oxo-5alpha-lanosta-8,24-dien-21-oic acid (10), 3alpha-acetoxy-5alpha-lanosta-8,24-dien-21-oic acid ester beta-d-glucoside (11), and tsugaric acid B (12), were assessed in vitro by determining their inhibitory effects on the chemical mediators released from mast cells, neutrophils, and macrophages. Compound 10 showed a significant inhibitory effect on the release of beta-glucuronidase from rat neutrophils stimulated with formyl-Met-Leu-Phe (fMLP)/cytochalasin B (CB) whereas compound 9 significantly inhibited superoxide anion formation in fMLP/CB-stimulated rat neutrophils. Compound 10 also exhibited a potent inhibitory effect on NO production in lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma)-stimulated N9 microglial cells. Moreover, compound 9 was also able to protect human keratinocytes against damage induced by ultraviolet B (UV B) light, which indicated 9 could protect keratinocytes from photodamage.