Hye-Eun Choi

Fucosterol isolated from Undaria pinnatifida inhibits lipopolysaccharide-induced production of nitric oxide and pro-inflammatory cytokines via the inactivation of nuclear factor-κB and p38 mitogen-activated protein kinase in RAW264.7 macrophages.

It has been reported that fucosterol has anti-diabetic, anti-oxidant, and anti-osteoporotic effects. We investigated the anti-inflammatory effects and the underlying molecular mechanism of fucosterol in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. Fucosterol suppressed the expressions of inducible nitric oxide synthase (iNOS), tumour necrosis factor-α (TNF-α), and interleukin-6 (IL-6) by downregulating their transcriptions, and subsequently inhibited the productions of nitric oxide, TNF-α, and IL-6. In addition, fucosterol attenuated LPS-induced DNA binding and the transcriptional activity of nuclear factor-κB (NF-κB). These reductions were accompanied by parallel reductions in the phosphorylation and nuclear translocation of NF-κB. Furthermore, fucosterol attenuated the phosphorylations of mitogen-activated protein kinase kinases 3/6 (MKK3/6) and mitogen-activated protein kinase-activated protein kinase 2 (MK2), which are both involved in the p38 MAPK pathway. These results suggest that the anti-inflammatory effects of fucosterol are associated with the suppression of the NF-κB and p38 MAPK pathways.

Roxatidine suppresses inflammatory responses via inhibition of NF‐κB and p38 MAPK activation in LPS‐induced RAW 264.7 macrophages

Roxatidine is a novel, specific, competitive H2‐receptor antagonist that is used to treat gastric and duodenal ulcers, and which is known to suppress the growth of several tumors by reducing vascular endothelial growth factor (VEGF) expression. Nevertheless, it remains unclear whether roxatidine has anti‐inflammatory effects. In this study, we the authors investigated the anti‐inflammatory effect of roxatidine in lipopolysaccharide (LPS)‐stimulated RAW 264.7 macrophage cells. It was found that roxatidine dose‐dependently inhibited the productions of prostaglandin E2 (PGE2), nitric oxide (NO), and histamine, and the protein and mRNA expressions of cyclooxygenase‐2 (COX‐2), inducible nitric oxide synthase (iNOS), and histidine decarboxylase (HDC). In addition, roxatidine reduced the productions and expressions of VEGF‐1 and pro‐inflammatory cytokines, including those of tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), and interleukin‐6 (IL‐6). Electrophoretic mobility shift assays (EMSA) and reporter gene assays revealed that treatment with roxatidine attenuated the LPS‐induced DNA‐binding and transcriptional activity of nuclear factor kappa B (NF‐κB). In addition, it was found that pretreatment with roxatidine significantly inhibited the nuclear translocations of the p65 and p50 subunits of NF‐κB, and these inhibitions were not found to be associated with decreases in the phosphorylation or degradation of inhibitory kappa B‐α (IκBα). Furthermore, roxatidine suppressed the phosphorylation of p38 MAP kinase, but not of IκB kinase‐α/β (IKKα/β), c‐Jun NH2‐terminal kinase (JNK), or extracellular signal‐regulated kinase (ERK). Taken together, these results indicate that the anti‐inflammatory properties of roxatidine in LPS‐treated RAW 264.7 macrophages are mediated by the inhibition of NF‐κB transcriptional activity and the p38 MAP kinase pathway. J. Cell. Biochem. 112: 3648–3659, 2011.

Antiviral activity of carnosic acid against respiratory syncytial virus

BackgroundHuman respiratory syncytial virus (hRSV) is a leading cause of severe lower respiratory infection and a major public health threat worldwide. To date, no vaccine or effective therapeutic agent has been developed. In a screen for potential therapeutic agents against hRSV, we discovered that an extract of Rosmarinus officinalis exerted a strong inhibitory effect against hRSV infection. Subsequent studies identified carnosic acid as a bioactive constituent responsible for anti-hRSV activity. Carnosic acid has been shown to exhibit potent antioxidant and anti-cancer activities. Anti-RSV activity of carnosic acid was further investigated in this study.MethodsEffects of extracts from various plants and subfractions from R. officinalis on hRSV replication were determined by microneutralization assay and plaque assay. Several constituents were isolated from ethyl acetate fraction of R. officinalis and their anti-RSV activities were assessed by plaque assay as well as reverse-transcription quantitative PCR to determine the synthesis of viral RNAs.ResultsAmong the tested bioactive constituents of R. officinalis, carnosic acid displayed the most potent anti-hRSV activity and was effective against both A- and B-type viruses. Carnosic acid efficiently suppressed the replication of hRSV in a concentration-dependent manner. Carnosic acid effectively suppressed viral gene expression without inducing type-I interferon production or affecting cell viability, suggesting that it may directly affect viral factors. A time course analysis showed that addition of carnosic acid 8 hours after infection still effectively blocked the expression of hRSV genes, further suggesting that carnosic acid directly inhibited the replication of hRSV.ConclusionsThe current study demonstrates that carnosic acid, a natural compound that has already been shown to be safe for human consumption, has anti-viral activity against hRSV, efficiently blocking the replication of this virus. Carnosic acid inhibited both A- and B- type hRSV, while it did not affect the replication of influenza A virus, suggesting that its antiviral activity is hRSV-specific. Collectively, this study suggests the need for further evaluation of carnosic acid as a potential treatment for hRSV.

Anti-inflammatory effect of a standardized triterpenoid-rich fraction isolated from Rubus coreanus on dextran sodium sulfate-induced acute colitis in mice and LPS-induced macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE Rubus coreanus Miquel (Rosaceae), the Korean black raspberry, has traditionally been used to treat inflammatory diseases including diarrhea, asthma, stomach ailment, and cancer. Although previous studies showed that the 19α-hydroxyursane-type triterpenoids isolated from Rubus coreanus exerted anti-inflammatory activities, their effects on ulcerative colitis and mode of action have not been explored. This study was designed to assess the anti-inflammatory effects and the molecular mechanisms involving19α-hydroxyursane-type triterpenoid-rich fraction from Rubus coreanus (TFRC) on a mice model of colitis and lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. MATERIALS AND METHODS Experimental colitis was induced by DSS for 7 days in ICR mice. Disease activity indices (DAI) took into account body weight, stool consistency, and gross bleeding. Histological changes and macrophage accumulation were observed by immunohistochemical analysis. Pro-inflammatory markers were determined using immunoassays, RT-PCR, and real time PCR. Signaling pathway involving nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) activation was determined by luciferase assay and Western blotting. RESULTS In DSS-induced colitis mice, TFRC improved DAIs and pathological characteristics including colon shortening and colonic epithelium injury. TFRC suppressed tissue levels of pro-inflammatory cytokines and reduced macrophage infiltration into colonic tissues. In LPS-induced RAW 264.7 macrophages, TFRC inhibited the production of NO, PGE2, and pro-inflammatory cytokines by down-regulating the activation of NF-κB and p38 MAPK signaling. CONCLUSION The study demonstrates that TFRC has potent anti-inflammatory effects on DSS-induced colonic injury and LPS-induced macrophage activation, and supports its possible therapeutic and preventive roles in colitis.

Inhibition of cellular proliferation and induction of apoptosis in human lung adenocarcinoma A549 cells by T-type calcium channel antagonist.

The anti-proliferative and apoptotic activities of new T-type calcium channel antagonist, 6e (BK10040) on human lung adenocarcinoma A549 cells were investigated. The MTT assay results indicated that BK10040 was cytotoxic against human lung adenocarcinoma (A549) and pancreatic cancer (MiaPaCa2) cells in a dose-dependent manner with IC50 of 2.25 and 0.93μM, respectively, which is ca. 2-fold more potent than lead compound KYS05090 despite of its decreased T-type calcium channel blockade. As a mode of action for cytotoxic effect of BK10040 on lung cancer (A549) cells, this cancer cell death was found to have the typical features of apoptosis, as evidenced by the accumulation of positive cells for annexin V. In addition, BK10040 triggered the activations of caspases 3 and 9, and the cleavages of poly (ADP-ribose) polymerase (PARP). Moreover, the treatment with z-VAD-fmk (a broad spectrum caspase inhibitor) significantly prevented BK10040-induced apoptosis. Based on these results, BK10040 may be used as a potential therapeutic agent for human lung cancer via the potent apoptotic activity.

Synthesis, biological evaluation, and docking analysis of a novel family of 1-methyl-1H-pyrrole-2,5-diones as highly potent and selective cyclooxygenase-2 (COX-2) inhibitors

As a continuous research for discovery of new COX-2 inhibitors, we present the simple chemical synthesis, in vitro biological screening, and molecular docking study of 1H-pyrrole-2,5-dione derivatives. New synthetic compounds were evaluated for the inhibitory activities on LPS-induced PGE2 production in RAW 264.7 macrophage cells as well as the COX-1 and COX-2 inhibitory potency. Among them, compound 9d (MPO-0029) was identified as more potent and selective COX-2 inhibitor [PGE2 IC50=8.7 nM, COX-2 IC50=6.0 nM; COX-2 selectivity index (SI)=>168] than celecoxib. Molecular docking experiments were further performed against COX-2 and COX-1 isozymes to determine their probable binding models. Results of molecular docking studies revealed that compound 9d (MPO-0029) has stronger binding interaction with COX-2 than with COX-1 isozyme, and provided successfully complementary theoretical support for the obtained experimental biological data.

BRN-103, a novel nicotinamide derivative, inhibits VEGF-induced angiogenesis and proliferation in human umbilical vein endothelial cells.

Anti-angiogenesis is regarded as an effective strategy for cancer treatment, and vascular endothelial growth factor (VEGF) plays a key role in the regulations of angiogenesis and vasculogenesis. In the present study, the authors synthesized five novel nicotinamide derivatives which structurally mimic the receptor tyrosine kinase inhibitor sunitinib and evaluated their anti-angiogenic effects. Transwell migration assays revealed that 2-(1-benzylpiperidin-4-yl) amino-N-(3-chlorophenyl) nicotinamide (BRN-103), among the five derivatives most potently inhibited VEGF-induced human umbilical vein endothelial cells (HUVECs). In addition, BRN-103 dose-dependently inhibited VEGF-induced migration, proliferation, and capillary-like tube formation of HUVECs and vessel sprouting from mouse aortic rings. To understand the molecular mechanisms responsible for these activities, the authors examined the effect of BRN-103 on VEGF signaling pathways in HUVECs. BRN-103 was found to suppress the VEGF-induced phosphorylation of VEGF receptor 2 (VEGR2) and the activations of AKT and eNOS. Taken together, these results suggest that BRN-103 inhibits VEGF-mediated angiogenesis signaling in human endothelial cells.

Synthesis of tricyclic fused coumarin sulfonates and their inhibitory effects on LPS-induced nitric oxide and PGE2 productions in RAW 264.7 macrophages.

The regulations of NO and PGE2 productions are research topics of interest in the field of antiinflammatory drug development. In the present study, a series of tricyclic fused coumarin sulfonate derivatives was synthesized and evaluated for their abilities to inhibit NO and PGE2 productions in LPS-induced RAW 264.7 macrophages. Among all the target compounds, compound 1g possessing p-(trifluoromethyl)phenyl and fused cycloheptane moieties showed the highest inhibitory effects on NO and PGE2 productions. Compound 1g not only inhibited COX-2 activity but also reduced expressions of COX-2 and iNOS. Furthermore, ADME profiling showed that compounds 1g, 1j, 1m, and 1n are estimated to be orally bioavailable.

In vitro cytotoxicity on human ovarian cancer cells by T-type calcium channel blockers

The growth inhibition of human cancer cells via T-type Ca(2+) channel blockade has been well known. Herein, a series of new 3,4-dihydroquinazoline derivatives were synthesized via a brief SAR study on KYS05090 template and evaluated for both T-type Ca(2+) channel (Cav3.1) blockade and cytotoxicity on three human ovarian cancer cells (SK-OV-3, A2780 and A2780-T). Most of compounds except 6i generally exhibited more potent cytotoxicity on SK-OV-3 than mibefradil as a positive control regardless of the degree of T-type channel blockade. In particular, eight compounds (KYS05090, 6a and 6c-6h) showing strong channel blockade exhibited almost equal and more potent cytotoxicity on A2780 when compared to mibefradil. On A2780-T paclitaxel-resistant human ovarian carcinoma, two compounds (KYS05090 and 6d) were 20-fold more active than mibefradil. With respect to cell cycle arrest effect on A2780 and A2780-T cells, KYS05090 induced large proportion of sub-G1 phase in the cell cycle progression of A2780 and A2780-T, meaning the induction of cancer cell death instead of cell cycle arrest via blocking T-type Ca(2+) channel. Among new analogues, compounds 6g and 6h induced cell cycle arrest at G1 phase of A2780 and A2780-T cells in dose-dependent manner and exhibited strong anti-proliferation effects of ovarian cancer cells by blocking T-type Ca(2+) channel. Furthermore, 6g and 6h possessing strong cytotoxic effects could induce apoptosis of A2780 cells, which was detected by confocal micrographs using DAPI staining.

Berberine Decreased Inducible Nitric Oxide Synthase mRNA Stability through Negative Regulation of Human Antigen R in Lipopolysaccharide-Induced Macrophages

Berberine, a major isoquinoline alkaloid found in medicinal herbs, has been reported to possess anti-inflammatory effects; however, the underlying mechanisms responsible for its actions are poorly understood. In the present study, we investigated the inhibitory effects of berberine and the molecular mechanisms involved in lipopolysaccharide (LPS)-treated RAW 264.7 and THP-1 macrophages and its effects in LPS-induced septic shock in mice. In both macrophage cell types, berberine inhibited the LPS-induced nitric oxide (NO) production and inducible NO synthase (iNOS) protein expression, but it had no effect on iNOS mRNA transcription. Suppression of LPS-induced iNOS protein expression by berberine occurred via a human antigen R (HuR)-mediated reduction of iNOS mRNA stability. Molecular data revealed that the suppression on the LPS-induced HuR binding to iNOS mRNA by berberine was accompanied by a reduction in nucleocytoplasmic HuR shuttling. Pretreatment with berberine reduced LPS-induced iNOS protein expression and the cytoplasmic translocation of HuR in liver tissues and increased the survival rate of mice with LPS-induced endotoxemia. These results show that the suppression of iNOS protein expression by berberine under LPS-induced inflammatory conditions is associated with a reduction in iNOS mRNA stability resulting from inhibition of the cytoplasmic translocation of HuR.