20(S)-Protopanaxatriol, one of ginsenoside metabolites, inhibits inducible nitric oxide synthase and cyclooxygenase-2 expressions through inactivation of nuclear factor-kappaB in RAW 264.7 macrophages stimulated with lipopolysaccharide.

Ginsenosides from Panax ginseng are metabolized by human intestinal bacteria after oral administration of ginseng extract. 20(S)-Protopanaxatriol (PPT) is one of the major metabolites of ginsenosides. Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are important enzymes that mediate inflammatory processes. Improper up-regulation of iNOS and/or COX-2 has been associated with the pathogenesis of inflammatory diseases and certain types of human cancers. Here, we investigated whether PPT could modulate iNOS and COX-2 expressions in RAW 264.7 macrophages stimulated with the endotoxin lipopolysaccharide (LPS). We found that PPT blocked the increase in LPS-induced iNOS and COX-2 expressions through inactivation of nuclear factor-kappaB by preventing I-kappaBalpha phosphorylation and degradation. Thus, it may be possible to develop PPT as a useful agent for chemoprevention of cancer or inflammatory diseases.

Synergistic cooperation between water-soluble chitosan oligomers and interferon-gamma for induction of nitric oxide synthesis and tumoricidal activity in murine peritoneal macrophages.

The effects of water-soluble chitosan oligomers (WSCO) on the synthesis of nitric oxide (NO) by murine peritoneal macrophages and on macrophage-mediated cytotoxicity towards murine fibrosarcoma Meth A cells were investigated. WSCO alone had no effect on NO synthesis and killing of tumor cells. However, treatment of macrophages with a combination of WSCO and interferon-gamma (IFN-gamma) synergically increased NO synthesis and enhanced killing of tumor cells. The synergism between IFN-gamma and WSCO in NO synthesis and tumoricidal activity was mainly dependent on increased secretion of tumor necrosis factor-alpha by WSCO.

In vitro anti-proliferative effect of 1,2,3,4,6-penta-O-galloyl-beta-D-glucose on human hepatocellular carcinoma cell line, SK-HEP-1 cells.

The root of Paeonia suffruticosa ANDREWS is an important Chinese crude drug used in many traditional prescriptions. 1,2,3,4,6-penta-O-galloyl-beta-D-glucose (PGG), a major component of this crude drug, was found to exhibit in vitro growth-inhibiting effect on human hepatocellular carcinoma cell line, SK-HEP-1 cells. The growth-inhibitory effect was related to the ability of PGG not only to cause a G(0)/G(1) phase arrest but also to suppress the activation of nuclear factor-kappa B. Neither apoptosis nor necrosis was observed in the cells treated with PGG. These findings suggest that PGG could be a candidate for developing a low-toxic anticancer agent.

Induction of granulocytic differentiation in acute promyelocytic leukemia cells (HL-60) by water-soluble chitosan oligomer.

Water-soluble chitosan oligomer (WSCO) has been reported to have anticancer activity, immuno-enhancing effect and antimicrobial activity. However, other biological activities are unknown. Herein, we have shown that WSCO is able to inhibit proliferation of human leukemia HL-60 cells and induce these cells to differentiate. Treatment with WSCO for 4 days resulted in a concentration-dependent reduction in HL-60 cell growth as measured by cell counting and MTT assay. This effect was accompanied by a marked increase in the proportion of G(0)/G(1) cells as measured by flow cytometry. WSCO also induced differentiation of the cells as measured by phorbol ester-dependent reduction of NBT, morphological changes as examined by Wright-Giemsa staining and expression of CD11b but not of CD14 as analysed by flow cytometry, indicating differentiation of HL-60 cells toward granulocyte-like cells. A combination of low dose of WSCO with all-trans retinoic acid, a differentiating agent toward granulocyte-like cells, exhibited a synergistic effect on the differentiation. In addition, treatment of HL-60 cells with WSCO for 6 or 8 days resulted in the induction of apoptosis as assayed qualitatively by agarose gel electrophoresis and quantitatively by Annexin V technique using flow cytometry. Collectively, there is a potential for WSCO in the treatment of myeloid leukemia.

TCDD-up-regulation of IGFBP-6 and IL-5R alpha subunit genes in vivo and in vitro.

Although the potent environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been well known for its immunosuppressive activity, the mechanisms of its action have been difficult to elucidate. This is partly due to its inability to exert its effects in vitro. To further elucidate the underlying mechanisms of TCDD effects, we screened for genes that are regulated by the in vivo TCDD treatment of mice that are challenged with allogeneic tumor cells. RNA, collected from lymphoid organs including the thymus, draining lymph nodes, and bone marrow, was reverse-transcribed to cDNA and hybridized to DNA arrays that consisted of 588 genes (ClonTech, USA). The expression of the NF-kappaB p65, c-jun, and p27(Kip1) genes was increased by the TCDD treatment, as previously reported. In addition, we found that the expression of several genes, which were not reported as regulated by TCDD, were modulated by TCDD. Some genes, including insulin-like growth factor-binding protein-6 (IGFBP-6) and IL-5R alpha, were upregulated; while other genes, including CD14, were down-regulated. The expression of the IGFBP-6 and IL-5R alpha subunit genes by TCDD in the thymus was confirmed by RT-PCR and Western blot analyses. Furthermore, TCDD effects on the expression of the IGFBP-6 gene was also observed with EL4 mouse thymoma cells. This suggests that IGFBP-6 may be involved in thymic atrophy, and EL4 cells may be used as an in vitro model for studying molecular mechanisms of thymic atrophy.

Penta-O-galloyl-beta-D-glucose inhibits phorbol myristate acetate-induced interleukin-8 [correction of intereukin-8] gene expression in human monocytic U937 cells through its inactivation of nuclear factor-kappaB.

We investigated the effects of the gallotannin penta-O-galloyl-beta-d-glucose (PGG) on interleukin (IL)-8 gene expression and nuclear factor (NF)-kappaB activation. PGG inhibited IL-8 production and gene expression in human monocytic U937 cells stimulated with phorbol myristate acetate (PMA), as measured by enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction analysis, respectively. PGG also inhibited PMA-mediated NF-kappaB activation, as measured by electromobility shift assay. Furthermore, PGG prevented PMA-mediated degradation of the NF-kappaB inhibitory protein I-kappaBalpha, as measured by Western blot analysis. PGG also inhibited both IL-8 production and NF-kappaB activation in the U937 cells stimulated with tumor necrosis factor-alpha. These results suggest that PGG, a major constituent of the root cortex of Paeonia suffruticosa ANDREWS, can inhibit IL-8 gene expression by a mechanism involving its inhibition of NF-kappaB activation, which is dependent on I-kappaBalpha degradation.

Capsazepine, a vanilloid receptor antagonist, inhibits the expression of inducible nitric oxide synthase gene in lipopolysaccharide-stimulated RAW264.7 macrophages through the inactivation of nuclear transcription factor-kappa B.

High amounts of nitric oxide (NO) production following the induction of inducible NO synthase (iNOS) gene expression has been implicated in the pathogenesis of inflammatory diseases. Capsaicin, a vanilloid receptor agonist, is known to have an inhibitory effect on NO production in macrophages. In the present study, we have found that capsazepine (CAPZ), a vanilloid receptor antagonist, also inhibited NO and iNOS protein syntheses induced by lipopolysaccharide in RAW264.7 macrophages via the suppression of iNOS mRNA. The mechanistic studies showed that CAPZ inhibited the expression of iNOS mRNA through the inactivation of nuclear transcription factor-kappa B (NF-kappa B). Thus, capsazepine may be a useful candidate for the development of a drug to treat inflammatory diseases related to iNOS gene overexpression.

Inhibition of TNF-alpha, IL-1beta, and IL-6 productions and NF-kappa B activation in lipopolysaccharide-activated RAW 264.7 macrophages by catalposide, an iridoid glycoside isolated from Catalpa ovata G. Don (Bignoniaceae).

Catalposide, the major iridoid glycoside isolated from the stem bark of Catalpa ovata G. Don (Bignoniaceae), was found to inhibit the productions of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-6 (IL-6), and the activation of nuclear factor kappaB (NF-kappaB) in RAW 264.7 macrophages activated with lipopolysaccharide (LPS). Catalposide also inhibited the expressions of TNF-alpha, IL-1beta, and IL-6 genes and the nuclear translocation of p65 subunit of NF-kappaB in LPS-activated RAW 264.7 cells. Flow cytometric analysis revealed that catalposide suppressed the binding of FITC-conjugated LPS to CD14 on the surface of cells, probably resulting in the inhibitory effects on TNF-alpha, IL-1beta, and IL-6 productions and NF-kappaB activation. These findings suggest that catalposide could be an attractive candidate for adjunctive therapy in gram-negative bacterial infections.

Induction of apoptosis by 4-acetyl-12,13-epoxyl-9-trichothecene-3,15-diol from Isaria japonica Yasuda through intracellular reactive oxygen species formation and caspase-3 activation in human leukemia HL-60 cells.

Recently we have reported that the trichothecene mycotoxin 4-acetyl-12,13-epoxyl-9-trichothecene-3,15-diol (AETD) from the fruiting bodies of Isaria japonica Yasuda is a potent inducer of apoptosis in human promyelocytic HL-60 cells. The present study aims to characterize the molecular events leading to AETD-induced apoptosis in HL-60 cells. The percentage of apoptotic cells (annexin-V-positive cell population) increased dose- and time-dependently after AETD exposure. Apoptosis of HL-60 cells by AETD was associated with the formation of intracellular reactive oxygen species (ROS), the depletion of intracellular glutathione (GSH) and the activation of caspase-3. Pretreating the cells with the antioxidant N-acetyl-L-cystein (NAC) and the caspase-3 inhibitor Z-DEVD-fmk abrogated AETD-induced apoptosis and caspase-3 activation. NAC blocked intracellular ROS formation and GSH depletion, but Z-DEVD-fmk did not. These results indicate that AETD induces apoptosis in HL-60 cells by causing intracellular ROS formation and GSH depletion followed by the downstream event of caspase-3 activation.

Inhibitory effects of methanol extract of Cyperus rotundus rhizomes on nitric oxide and superoxide productions by murine macrophage cell line, RAW 264.7 cells.

The rhizomes of Cyperus rotundus (C. rotundus) have been used in oriental traditional medicines for the treatment of stomach and bowel disorders, and inflammatory diseases. Nitric oxide (NO) and superoxide (O2-) are important mediators in the pathogenesis of inflammatory diseases. This study was undertaken to address whether the metanol (MeOH) extract of rhizomes of C. rotundus could modulate NO and O2- productions by murine macrophage cell line, RAW 264.7 cells. The MeOH extract of rhizomes of C. rotundus showed the inhibition of NO production in a dose-dependent manner by RAW 264.7 cells stimulated with interferon-gamma plus lipopolysaccharide. The inhibition of NO production by the extract was due to the suppression of iNOS protein, as well as iNOS mRNA expression, determined by Western and Northern blotting analyses, respectively. In addition, the MeOH extract suppressed the production of O2- by phorbol ester-stimulated RAW 264.7 cells in dose- and time-dependent manners. Collectively, these results suggest that the MeOH extract of rhizomes of C. rotundus could be developed as anti-inflammatory candidate for the treatment of inflammatory diseases mediated by overproduction of NO and O2-.

The aqueous extract of Rhodiola sachalinensis root enhances the expression of inducible nitric oxide synthase gene in RAW264.7 macrophages.

In the present study, we examined the effects of the aqueous extract of Rhodiola sachalinensis root (RSE) on the expression of inducible nitric oxide (NO) synthase (iNOS) gene in RAW264.7 macrophages. RSE synergistically increased NO synthesis in interferon-gamma-primed macrophages. Reverse transcriptase polymerase chain reaction and Northern blotting analysis revealed that RSE may provide a second triggering signal for the synergistic induction of iNOS mRNA expression. Thus, iNOS-mediated NO synthesis in response to RSE may be one mechanism whereby this herbal medicine elicits its therapeutic effects.

Inhibitory effects of the root cortex of Paeonia suffruticosa on interleukin-8 and macrophage chemoattractant protein-1 secretions in U937 cells.

In an effort to elucidate the mechanism of the anti-inflammatory effect of mudanpi, the root cortex of Paeonia suffruticosa Andrews (Ranunculaceae), we determined the effects of the methanolic extract of mudanpi (MEM) on the secretions of interleukin (IL)-8, a major mediator of acute neutrophil-mediated inflammation, and macrophage chemoattractant protein (MCP)-1, a major mediator of chronic macrophage-mediated inflammation, in human monocytic U937 cells stimulated with phorbol myristate acetate (PMA). MEM significantly inhibited PMA-induced secretions of IL-8 and MCP-1 proteins in a dose-dependent manner. The inhibition of these chemokines by MEM was due to its suppression of IL-8 and MCP-1 genes. In addition, 1,2,3,4,6-penta-O-galloyl-beta-D-glucose, one of major constituents isolated from MEM, inhibited PMA-induced secretions of IL-8 and MCP-1 proteins by its suppression of IL-8 and MCP-1 genes. Thus, one possible anti-inflammatory mechanism of mudanpi, an anti-inflammatory Chinese crude drug, may be to inhibit the secretions of inflammatory chemokines.

Inhibitory effects of the stem bark of Catalpa ovata G. Don. (Bignoniaceae) on the productions of tumor necrosis factor-alpha and nitric oxide by the lipopolisaccharide-stimulated RAW 264.7 macrophages.

In order to validate the use of the stem bark of Catalpa ovata G. Don. (Bignoniaceae) as an anti-inflammatory drug in the traditional Korean medicine, we have investigated the effects of the methanol extract of this folk medicine on the productions of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) on RAW 264.7 macrophages activated with the endotoxin lipopolysaccharide. The extract inhibited the productions of TNF-alpha and NO with significant decreases in mRNA levels of TNF-alpha and inducible NO synthase, suggesting that the stem bark of Catalpa ovata may have therapeutic potential in the control of inflammatory disorders.

Shear bond strength of repaired composite resins using a hybrid composite resin.

The shear bond strength of different types of composite resins repaired with a hybrid composite was evaluated. The hybrid composite resin was repaired with itself as a control. The results of this study showed that the shear bond strength of the repair with most types of composite resins was minimally adequate.

Ethyl acetate extract of the stem bark of Cudrania tricuspidata induces apoptosis in human leukemia HL-60 cells.

Apoptosis is now widely accepted as playing a role in tumorigenesis. An effective compound which can kill tumors via apoptotic pathway appears to be a relevant strategy to suppress various human tumors. The ethyl acetate extract from the stem bark of Cudrania tricuspidata (EACT) showed dose- and time-dependent cytotoxic effects on human leukemia HL-60 cells. DNA fragmentation and morphological changes, accompanied by condensed and fragmented nuclei, were observed in the cells cultured for 6 hr with EACT. These results suggest that the cytotoxicity of the crude extract from Cudrania tricuspidata against HL-60 cells is due to apoptosis.

Augmentation of NAD(+) by NQO1 attenuates cisplatin-mediated hearing impairment.

Cisplatin (cis-diaminedichloroplatinum-II) is an extensively used chemotherapeutic agent, and one of its most adverse effects is ototoxicity. A number of studies have demonstrated that these effects are related to oxidative stress and DNA damage. However, the precise mechanism underlying cisplatin-associated ototoxicity is still unclear. The cofactor nicotinamide adenine dinucleotide (NAD(+)) has emerged as a key regulator of cellular energy metabolism and homeostasis. Here, we demonstrate for the first time that, in cisplatin-mediated ototoxicity, the levels and activities of SIRT1 are suppressed by the reduction of intracellular NAD(+) levels. We provide evidence that the decrease in SIRT1 activity and expression facilitated by increasing poly(ADP-ribose) transferase (PARP)-1 activation and microRNA-34a through p53 activation aggravates cisplatin-mediated ototoxicity. Moreover, we show that the induction of cellular NAD(+) levels using ß-lapachone (ß-Lap), whose intracellular target is NQO1, prevents the toxic effects of cisplatin through the regulation of PARP-1 and SIRT1 activity. These results suggest that direct modulation of cellular NAD(+) levels by pharmacological agents could be a promising therapeutic approach for protection from cisplatin-induced ototoxicity.

Spatial genetic structure in a Neolitsea sericea population (Lauraceae).

Neolitsea sericea (Bl.) Koidz. (Lauraceae) is a dioecious, insect-pollinated, and broad-leaved evergreen tree with bird-dispersed seeds. We used allozyme loci, F-statistics, and spatial autocorrelation statistics (Moran's I ) to examine the changes in genetic structure among five age classes within a study population (60 m x 100 m area) in southern Korea. No significant differences in expected heterozygosity were found among the age classes. The mean F-values averaged over loci were similar among age classes and showed overall conformance of heterozygosities with Hardy-Weinberg proportions. Differences in allelic frequencies among age classes were small (mean G(ST)=0.012), and statistically significant only for one locus (Pgd-2). The mean Moran's I-values for each of five age classes indicated essentially random spatial distribution. The homogeneity of genetic structure and genetic diversity among the five age classes may reflect the occurrence of similar reproductive events, year after year. The results may reflect the attractive red drupes of N. sericea in that they cause various frugivorous birds to disperse the seed long distances and independently, which in turn may help N. sericea maintain higher levels of genetic diversity within populations.

Inhibitory effect of ethyl acetate fraction from Cudrania tricuspidata on the expression of nitric oxide synthase gene in RAW 264.7 macrophages stimulated with interferon-gamma and lipopolysaccharide.

It was found that the production of nitric oxide (NO) by RAW 264.7 macrophages stimulated with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) could be markedly inhibited by the ethyl-acetate-soluble fraction of 80% aqueous methanolic extract of stem barks of Cudrania tricuspidata (EACT). Inhibition of NO production was achieved by reducing inducible nitric oxide synthase (iNOS) expression at protein and mRNA levels and by inactivating nuclear factor-kappa B (NF-kappa B), but not by inhibiting iNOS activity. Thus, further phytochemical and pharmacological studies may lead to isolation and structural identification of an inhibitor of iNOS from C. tricuspidata, which has been used as a traditional medicine for curing inflammation.

Mistletoe lectin synergizes with paclitaxel in human SK-hep1 hepatocarcinoma cells.

The effect of mistletoe lectin I (ML-I), an inhibitor of ribosomal protein synthesis, on the in vitro cytotoxicity of a clinically important anticancer drug, paclitaxel, was studied on cultured human hepatocarcinoma SK-Hep1 cells using the microculture tetrazolium test. The interaction between these two agents was analyzed for true synergism using the ED50 isobologram. Synergism was observed in the simultaneous treatment of the cells with ML-I in combination with paclitaxel. In addition, 24-h exposure of the cells to a non-toxic dose of ML-I and lower toxic doses of paclitaxel in combination resulted in apoptotic cell death, as observed by agarose-gel electrophoresis of low-molecular-weight DNA and DNA flow cytometry. Thus, the results presented here indicate the potential clinical usefulness of ML-I combination therapy with paclitaxel.