In vitro cytotoxicity of Mokko lactone in human leukemia HL-60 cells: induction of apoptotic cell death by mitochondrial membrane potential collapse.

We studied the effect of mokko lactone (ML) isolated from the roots of Saussurea lappa (Compositae), a plant that is used for medicinal purposes in Korea, on the induction of apoptosis in human leukemia HL-60 cells. ML was cytotoxic to HL-60 cells, and this cytotoxic effect of ML appears to be attributable to its induction of apoptotic cell death, as ML induced nuclear morphologic changes and internucleosomal DNA fragmentation and increased the proportion of Annexin V-positive cells and the activity of caspase-3. Further studies revealed that the induction of apoptosis by ML was associated with the loss of mitochondrial membrane potential. Collectively, our results suggest that apoptosis induced by ML in HL-60 cells was executed by a collapse of mitochondrial membrane potential followed by the activation of caspase-3. This is the first report on the mechanism of apoptosis-inducing effect of ML.

Dehydrocostus lactone enhances tumor necrosis factor-alpha-induced apoptosis of human leukemia HL-60 cells.

Sesquiterpene lactones have raised considerable interest because of their ability to block the activation of nuclear transcription factor-kappaB (NF-kappaB). NF-kappaB plays an important role in the resistance of cancer cells to the induction of apoptosis by anticancer drugs and tumor necrosis factor-alpha (TNF-alpha). Pharmacological inhibition of NF-kappaB offers the promise of enhancing the efficacy of anticancer therapies. Here, we demonstrate that dehydrocostus lactone (DL), the major sesquiterpene lactone isolated from the roots of Saussurea lappa, inhibits NF-kappaB activation by preventing TNF-alpha-induced degradation and phosphorylation of its inhibitory protein I-kappaB alpha in human leukemia HL-60 cells and that DL renders HL-60 cells susceptible to TNF-alpha-induced apoptosis by enhancing caspase-8 and caspase-3 activities.

Hepatoprotective effect of baicalin, a major flavone from Scutellaria radix, on acetaminophen-induced liver injury in mice.

The protective effects of baicalin (BA), a major flavone from Scutellaria radix, on acetaminophen (AP)-induced hepatotoxicity and the possible mechanism(s) of its protective action were investigated in mice. Treatment with BA (300 mg/kg, p.o.) 0.5 h after AP administration significantly prevented an increase in plasma alanine aminotransferase and aspartate aminotransferase activities and AP-induced hepatic necrosis, and also reduced AP-induced mortality from 43% to 0%. In addition, oral treatment with BA significantly prevented AP-induced depletion of glutathione (GSH) contents. However, BA treatment, by itself, did not affect hepatic GSH contents. The effect of BA on the cytochrome P450 2E1 (CYP2E1), the major isozyme involved in AP bioactivation, was investigated. Oral treatment of mice with BA resulted in a significant decrease in AP-induced CYP2E1 activity together with its inhibition of AP-induced CYP2EI expression. These results show that the hepatoprotective effects of BA against AP overdose may be due to its ability to block the bioactivation of AP by inhibiting CYP2E1 expression.

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.

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.

Inhibitory effect of retinoic acid on expression of inducible nitric oxide synthase gene in l929 cells.

Inflammation has been known to be associated with excess synthesis of nitric oxide (NO) by inducible NO synthase (iNOS). Retinoids have been reported to have anti-inflammatory activity, but the mechanism by which they can elicit this activity is poorly understood. The effects of retinoids on NO synthesis and iNOS gene expression in murine fibroblast L929 cells were examined. Treatment of the cells with interferon-y resulted in excess NO synthesis and iNOS gene expression. All-trans-retinoic acid significantly inhibited NO synthesis and iNOS gene expression in a dose-dependent manner. Similarly, 9-cis-retinoic acid also inhibited NO synthesis, but retinol did not show any inhibitory effect on NO synthesis. These findings suggest that the modulation of iNOS gene expression is another possible pathway by which retinoids may function as anti-inflammatory agents.

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.

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.

Prunioside A: a new terpene glycoside from Spiraea prunifolia.

Prunioside A (1) has been isolated from an EtOAc-soluble extract of Spiraea prunifolia var. simpliciflora by a combination of chromatographic techniques. The structure was determined primarily by extensive NMR experiments. Compound 1 is a unique terpene glycoside. Its acetylated derivative (1a) inhibited nitric oxide production in murine macrophage-like RAW 264.7 cells in a dose-dependent manner.

4-Acetyl-12,13-epoxyl-9-trichothecene-3,15-diol isolated from the fruiting bodies of Isariajaponica Yasuda induces apoptosis of human leukemia cells (HL-60).

The fruiting bodies of Isaria fungi have been traditionally used in Korea to treat cancer. An apoptosis-inducing compound, 4-acetyl-12,13-epoxyl-9-trichothecene-3,15-diol, was isolated from the methanol extract of fruiting bodies of Isaria japonica Yasuda by bioassay-guided fractionation. The apoptosis of the human leukemia cells (HL-60) by the compound was accessed by propidium iodide-staining flow cytometric analysis, and apoptosis-inducing activity at IC50 concentration (10 nmol/l) was further confirmed by a nuclear morphological change, a ladder pattern of internucleosomal DNA fragmentation, and an activation of caspase-3.

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.

Rhodiola sachalinesis induces the expression of inducible nitric oxide synthase gene by murine fetal hepatocytes (BNL CL.2).

We have examined the effect of the aqueous extract of Rhodiola sachalinensis root (RSE), a traditional herbal medicine, on nitric oxide (NO) synthesis in murine fetal hepatocytes (BNL CL.2) by measuring the stable end-product nitrite and the mRNA of inducible NO synthase (iNOS). Interferon-gamma (IFN-gamma) by itself failed to induce NO synthesis in BNL CL.2 cells. RSE also did not elicit NO synthesis at concentrations up to 1,000 microg/ml, but dose- and time-dependently induced NO synthesis in the presence of IFN-gamma in BNL CL.2 cells. Whereas RSE or IFN-gamma failed to induce detectable levels of iNOS mRNA, a combination of RSE and IFN-gamma markedly induced iNOS mRNA in BNL CL.2 cells. Thus, we found that RSE triggered IFN-gamma-primed BNL CL.2 cells to synthesize NO by inducing iNOS gene expression. The capability of RSE to induce NO synthesis might be related to the therapeutic efficacy of RSE on the liver diseases.

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.

Tenellic acids A-D: new bioactive diphenyl ether derivatives from the aquatic fungus Dendrospora tenella.

Tenellic acids A-D (1-4) were isolated from cultures of the freshwater fungus Dendrospora tenella. The structures of these new diphenyl ether derivatives were determined primarily by analysis of NMR data. Compounds 1-4 display antimicrobial activity against Gram-positive bacteria.