Kang Ro Lee

Suppression of inducible nitric oxide synthase and cyclooxygenase-2 expression in RAW 264.7 macrophages by sesquiterpene lactones.

The molecular mechanism underlying the suppression of lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma)-induced nitric oxide (NO) and prostaglandin (PG) E(2) production was investigated in RAW 264.7 macrophages treated with sesquiterpene lactones, zaluzanin-C and estafiatone, isolated from Ainsliaea. Zaluzanin-C and estafiatone decreased NO production in LPS/IFN-gamma-stimulated RAW 264.7 macrophages with an IC50 of about 6.61 microM and 3.80 microM, respectively. In addition, these compounds inhibited the synthesis of PGE(2) in LPS/IFN-gamma-treated RAW 264.7 macrophages. Furthermore, treatment with zaluzanin-C and estafiatone resulted in a decrease in inducible No Synthase (iNOS) and Cyclooxygenase-2 (COX-2) protein and mRNA expression levels. Zaluzanin-C and estafiatone inhibited nuclear factor-kappaB (NF-kappaB) activation, a transcription factor necessary for iNOS and COX-2 expression in response to LPS/IFN-gamma. This effect was accompanied by parallel reduction of phosphorylation and degradation of inhibitor of kappaB (IkB). In addition, these effects were completely blocked by treatment with cysteine, indicating that the inhibitory effect of zaluzanin-C and estafiatone might be mediated by alkylation of either NF-kappaB itself or an upstream molecule of NF-kappaB. These results demonstrate that the suppression of NF-kappaB activation by zaluzanin-C and estafiatone might be attributed to inhibition of nuclear translocation of NF-kappaB resulting from blockade of the degradation of IkappaB, leading to suppression of the expression of iNOS and COX-2, which play important roles in inflammatory signaling pathways.

Antioxidative flavonoids from the leaves ofMorus alba

Nine flavonoids (1–9) were isolated from the leaves ofMorus alba (Moraceae). The structures of compounds were determined to be kaempferol-3-O-β-D-glucopyranoside (astragalin,1) kaempferol-3-O-(6″-O-acetyl)-β-D-glucopyranoside (2), quercetin-3-O-(6″-O-acetyl)-β-D-glucopyranoside (3), quercetin-3-O-β-D-glucopyranoside (4), kaempferol-3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside (5), quercetin-3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside (rutin,6), quercetin-3-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside (7), quercetin-3,7-di-O-β-D-glucopyranoside (8) and quercetin (9) on the basis of spectroscopic and chemical studies. Compounds7 and9 exhibited significant radical scavenging effect on 1,1-diphenyl-2-picryl-hydrazyl radical.

Suppression of inducible nitric oxide synthase expression in RAW 264.7 macrophages by two β-carboline alkaloids extracted from Melia azedarach

We investigated the mechanism of suppression of inducible nitric oxide synthase (iNOS) by two beta-carboline alkaloids isolated from Melia azedarach, 4,8-dimethoxy-1-vinyl-beta-carboline (compound 1, C-1) and 4-methoxy-1-vinyl-beta-carboline (compound 2, C-2). iNOS activity in a cell-free extract of lipopolysaccharide/interferon-gamma-stimulated RAW 264.7 cells was found to be markedly increased, and this increase was prevented by C-1 and C-2, accompanied by the parallel reduction in nitrite accumulation in culture medium. However, C-1 and C-2 had no further effect on the iNOS activity prepared from fully lipopolysaccharide/interferon-gamma-stimulated RAW 264.7 cells. Treatment with C-1 or C-2 decreased the levels of iNOS protein and mRNA in a concentration-dependent manner. In addition, prostaglandin E(2) production, cyclooxygenase-2 protein and DNA binding of nuclear factor-kappaB (NF-kappaB) in lipopolysaccharide-stimulated RAW 264.7 cells were reduced by these compounds. These results indicate that C-1 and C-2 primarily inhibit iNOS and cyclooxygenase-2 activities via the suppression of de novo synthesis of these two enzymes, and that the inhibition of iNOS expression may be associated with the inhibition of NF-kappaB activation. Taken together, the results suggest that suppression of iNOS and cyclooxygenase-2 induction by lipopolysaccharide is responsible for the anti-inflammatory activity of these alkaloids through selective inhibition of the expression of genes, which play important roles in inflammatory signaling pathways.

Ergolide, sesquiterpene lactone from Inula britannica, inhibits inducible nitric oxide synthase and cyclo-oxygenase-2 expression in RAW 264.7 macrophages through the inactivation of NF-κB

We investigated the mechanism of suppression of inducible nitric oxide synthase (iNOS) and cyclo‐oxygenase‐2 (COX‐2) by ergolide, sesquiterpene lactone from Inula britannica. iNOS activity in cell‐free extract of LPS/IFN‐γ‐stimulated RAW 264.7 macrophages was markedly attenuated by the treatment with ergolide. Its inhibitory effect on iNOS was paralleled by decrease in nitrite accumulation in culture medium of LPS/IFN‐γ‐stimulated RAW 264.7 macrophages in a concentration‐dependent manner. However, its inhibitory effect does not result from direct inhibition of the catalytic activity of NOS. Ergolide markedly decreased the production of prostaglandin E2 (PGE2) in cell‐free extract of LPS/IFN‐γ‐stimulated RAW 264.7 macrophages in a concentration‐dependent manner, without alteration of the catalytic activity of COX‐2 itself. Ergolide decreased the level of iNOS and COX‐2 protein, and iNOS mRNA caused by stimulation of LPS/IFN‐γ in a concentration‐dependent manner, as measured by Western blot and Northern blot analysis, respectively. Ergolide inhibited nuclear factor‐κB (NF‐κB) activation, a transcription factor necessary for iNOS and COX‐2 expression in response to LPS/IFN‐γ. This effect was accompanied by the parallel reduction of nuclear translocation of subunit p65 of NF‐κB as well as IκB‐α degradation. In addition, these effects were completely blocked by treatment of cysteine, indicating that this inhibitory effect of ergolide could be mediated by alkylation of NF‐κB itself or an upstream molecule of NF‐κB. Ergolide also directly inhibited the DNA‐binding activity of active NF‐κB in LPS/IFN‐γ‐pretreated RAW 264.7 macrophages. These results demonstrate that the suppression of NF‐κB activation by ergolide might be attributed to the inhibition of nuclear translocation of NF‐κB resulted from blockade of the degradation of IκB and the direct modification of active NF‐κB, leading to the suppression of the expression of iNOS and COX‐2, which play important roles in inflammatory signalling pathway.

Suppression by a sesquiterpene lactone from Carpesium divaricatum of inducible nitric oxide synthase by inhibiting nuclear factor-κB activation

Excessive nitric oxide (NO) produced by inducible NO synthase (iNOS) acts as a causative regulator in various inflammatory disease states. Carpesium divaricatum has been used in Korean traditional herbal medicine for its antipyretic, analgesic, vermifugic, and anti-inflammatory properties. We investigated the molecular mechanism for the suppression of lipopolysaccharide/interferon-gamma (LPS/IFN-gamma)-induced NO production in RAW 264.7 macrophages by the sesquiterpene lactone 2beta,5-epoxy-5,10-dihydroxy-6alpha-angeloyloxy-9beta-isobutyloxy-germacran-8alpha,12-olide (C-1), which has been identified recently as a new compound from C. divaricatum. C-1 decreased NO production in LPS/IFN-gamma-stimulated RAW 264.7 cells in a concentration-dependent manner, with an IC50 of approximately 2.16 microM; however, it had no direct effect on the iNOS activity of fully LPS/IFN-gamma-stimulated RAW 264.7 cells. Furthermore, treatment with C-1 led to a decrease in iNOS protein and mRNA. These effects appear to be due to inhibition of nuclear factor-kappaB (NF-kappaB) activation through a mechanism involving stabilization of the NF-kappaB/inhibitor of the kappaB (I-kappaB) complex, since inhibition of NF-kappaB DNA binding activity by C-1 was accompanied by a parallel reduction of nuclear translocation of subunit p65 of NF-kappaB and I-kappaBalpha degradation. Taken together, the results suggest that the ability of C-1 to inhibit iNOS gene expression may be responsible, in part, for its anti-inflammatory effects.

Antitumor and immunomodulating activities of the polysaccharide fractions fromArtemisia selengensis andArtemisia iwayomogi

Effects of the polysaccharide fractions purified fromArtemisia selengensis andArtemisia iwayomogi on the immune system was studied. The polysaccharide fractions, respectively called ASP1 and AIP1, may interact with macrophages and lymphocytes in spleen, increasing the population of those cell typesin vivo andin vitro. Both ASP1 and AIP1 fractions also suppress transplanted tumor cell growth and augment antibody production. This study suggests that ASP1 and AIP1 fractions may have immunomodulating and antitumor activities.

Apoptotic potential of sesquiterpene lactone ergolide through the inhibition of NF‐κB signaling pathway

Treatment with ergolide, a sesquiterpene lactone from Inula britannica var chinensis, caused the induction of apoptosis in Jurkat T cells, which was confirmed by DNA fragmentation, caspase‐3 activation and cleavage of poly(ADP‐ribose) polymerase in response to ergolide. Furthermore, mitochondrial dysfunction appeared to be associated with ergolide‐induced apoptosis, because Bax translocation and cytochrome c release were stimulated by ergolide. In parallel, the nuclear factor‐κB (NF‐κB) signaling pathway was significantly inhibited by ergolide, which was accompanied by down‐regulation of cell survival molecules, such as X‐chromosome‐linked inhibitor of apoptosis and Bcl‐2. In addition, the JNK signaling pathway was involved in ergolide‐induced apoptosis. Collectively, our results identified a new mechanism for the anti‐cancer property of ergolide, attributable to the induction of apoptosis through down‐regulation of cell survival signal molecules resulting from inhibition of the NF‐κB signaling pathway.

β-Carboline Alkaloid Suppresses NF-κB Transcriptional Activity Through Inhibition of IKK Signaling Pathway

Nuclear factor (NF)-κB transcription factors play an evolutionarily conserved and critical role in the triggering and coordination of both innate and adaptive immune responses. Therefore, there is intense interest in understanding the regulation of this transcription factor in the context of various diseases. Studies investigated the suppression mechanism of NF-κB signaling pathways by a β-carboline alkaloid (C-1) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. β-Carboline alkaloid decreased the level of inducible nitric oxide sythase (iNOS) protein and NOS promoter activities in a concentration-dependent manner. This effect was accompanied by the reduction of NF-κB DNA binding activity as well as NF-κB nuclear translocation. In addition, β-carboline alkaloid reduced the degradation and phosphorylation of IκB, and attenuated IKK activity in LPS-stimulated RAW 264.7 cells. Taken together, these results indicate that β-carboline alkaloid has the capability to suppress NF-κB signaling pathway through...

A new cytotoxic acyclic diterpene from Carpesium divaricatum.

A new acyclic diterpene (1) and a known acyclic diterpene, 12(S)-hydroxygeranylgeraniol (2) were isolated from the aerial parts ofCarpesium divaricatum. The structure of1 was determined to be (2E, 10E)-1, 12-dihydroxy-18-acetoxy-3,7,15-trimethylhexadeca-2,10,14-triene (1) on the basis of spectroscopic studies. Compounds1 and2 exhibited cytotoxicity against cultured human tumor cell lines, A549, SK-OV-3, SK-MEL-2, XF498, and HCT15, with ED50 values ranging from 4.3–10.2 μg/ml and 4.1–8.3 μg/ml, respectively.

Stability and cytotoxicity of Fab-ricin A immunotoxins prepared with water soluble long chain heterobifunctional crosslinking agents

The effects of the hindered and non-hindered water soluble long-chain disulfide bonds on the stability and cytotoxicity of the ricin A chain (RTA) immunotoxin were examined. The RTA immunotoxins were prepared with the Fab fragments of anti-common acute lymphoblastic leukemia antigen (CALLA) monoclonal antibody (Fab-RTA) using sulfosuccinimidyl-6-[(-methyl-(-(2-pyridyldithio)toluamido] hexanoate (S-LC-SMPT) and sulfosuccinimidyl-6-[3-(2-pyridyldi-thio)-propionamido]hexanoate (S-LC-SPDP). The prepared Fab-RTA immunotoxins were evaluated for their conjugation yield, immunoreactivity, thermal and disulfide bond stability and cytotoxicity. The conjugation yield of the Fab-RTA immunotoxin from the water soluble long chain crosslinking agents, S-LC-SMPT and S-LC-SPDP, were comparable. Both Fab-RTA immunotoxins exhibited a similar immunoreactivity and thermal stability in aqueous solution. However, S-LC-SMPT mediated Fab-RTA, sterically hindered, showed an enhanced disulfide bond stability in vitro over S-LC-SPDP mediated one. In the cytotoxicity against antigenic cell Daudi, the S-LC-SMPT mediated RTA immunotoxin maintained a comparable cytotoxicity, compared with S-LC-SPDP mediated Fab-RTA immunotoxin.