[show abstract][hide abstract] ABSTRACT: The biochemical mechanisms of cell death by oleifolioside B (OB), a cycloartane-type triterpene glycoside isolated from Dendropanax morbifera Leveille, were investigated in A549 human lung carcinoma cells. Our data indicated that exposure to OB led to caspase activation and typical features of apoptosis; however, apoptotic cell death was not prevented by z-VAD-fmk, a pan-caspase inhibitor, demonstrating that OB-induced apoptosis was independent of caspase activation. Subsequently, we found that OB increased autophagy, as indicated by an increase in monodansylcadaverine fluorescent dye-labeled autophagosome formation and in the levels of the autophagic form of microtubule-associated protein 1 light chain 3 and Atg3, an autophagy-specific gene, which is associated with inhibiting phospho-nuclear factor erythroid 2-related factor 2 (Nrf2) expression. However, pretreatment with bafilomycin A1, an autophagy inhibitor, attenuated OB-induced apoptosis and dephosphorylation of Nrf2. The data suggest that OB-induced autophagy functions as a death mechanism in A549 cells and OB has potential as a novel anticancer agent capable of targeting apoptotic and autophagic cell death and the Nrf2 signaling pathway.
International Journal of Oncology 12/2013; 43(6):1943-50. · 2.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: In the present study, we firstly found that cordycepin elevated the gene expression of the human GD3 synthase (hST8Sia I) in human neuroblastoma SK-N-BE(2)-C cells. To elucidate the mechanism underlying the upregulation of hST8Sia I gene expression in cordycepin-treated SK-N-BE(2)-C cells, functional characterization of the promoter region of the hST8Sia I gene was performed. Analysis of promoter activity using varying lengths of 5'-flanking region showed a dramatic increase by cordycepin in the -1146 to -646 region, which contains putative binding sites for transcription factors c-Ets-1, CREB, AP-1, and NF-κB. Site-directed mutagenesis for these binding sites and chromatin immunoprecipitation assay revealed that the NF-κB binding site at -731 to -722 is essential for the cordycepin-induced expression of the hST8Sia I in SK-N-BE(2)-C cells. Moreover, the hST8Sia I expression induced by cordycepin was significantly repressed by pyrrolidinedithiocarbamate, an inhibitor of NF-κB. These results suggested that cordycepin induces upregulation of hST8Sia I gene expression through NF-κB activation in SK-N-BE(2)-C cells.
Acta Biochimica et Biophysica Sinica 11/2013; · 1.81 Impact Factor
[show abstract][hide abstract] ABSTRACT: Triptolide (TPL) has been shown to inhibit cell proliferation and induce apoptosis in various human cancer cells; however, the precise mechanism of apoptosis induced by TPL in human melanoma cells has not yet been elucidated. In this study, we investigated the precise mechanism underlying cytocidal effects of TPL on human melanoma cells. Treatment of human melanoma cells with TPL significantly inhibited cell growth and induced apoptosis, as evidenced by flow cytometry and annexin V-fluorescein isothiocyanate analyses. TPL increased the levels of Fas and Fas-associated death domain (FADD) and induced cleavage of Bid by activation of caspase-8 and cytochrome c release from mitochondria to the cytosol, which resulted in activation of caspase-9 and caspase-3. Moreover, TPL-induced apoptosis in SK-MEL-2 cells was mediated through dephosphorylation of focal adhesion kinase (FAK) and its cleavage by caspase-8-mediated caspase-3 activation via upregulation of Fas expression. We also found that TPL mediated the dissociation of receptor-interacting protein (RIP) from FAK and enhanced the formation of RIP/Fas complex formation initiating cell death. In conclusion, our data firstly demonstrated that TPL induces apoptosis by both extrinsic and intrinsic apoptosis pathways in human melanoma cells and identified that RIP shuttles between Fas and FAK to mediate apoptosis.
Evidence-based Complementary and Alternative Medicine 05/2013; 2013. · 1.72 Impact Factor
[show abstract][hide abstract] ABSTRACT: An 80% ethanol extract of Hizikia fusiforme was obtained and followed by successive fractionation using the organic solvents n-hexane, ethyl acetate, and n-butanol to identify the antioxidative substance. The aqueous part of the nbutanol fractionation step, showing high antioxidative activity, was subjected to reverse-phase liquid chromatography. As a result, a substance purified from a BB-2 fraction showed high antioxidative activity. The m/z 419 [M+H] molecular ion peak in the fraction was observed by the analysis of the ESI-LC/MS spectrum. By the analysis of 1H NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) spectra, a unique compound of the fraction was biochemically identified as a 5-hydroxy-3,6,7,8,3´,4´- hexamethoxyflavone (5HHMF). We also investigated the effect of 5HHMF on human gastric AGS carcinoma cells. Western blot analysis suggested that the flavone substantially increased the levels of the death receptor-associated apoptosis mediators Fas, Fas L, FADD, TRADD, and DR4 in a concentration-dependent manner. The levels of Fas, Fas L, TRADD, and DR4 in the cells treated with 5HHMF (5 microgram/ml) were approximately 26.4-, 12.8-, 6.7-, and 9.8- times higher than those of non-treated cells, respectively. Of note, the level of FADD protein in the cells exposed to 5HHMF (1 microgram/ml) increased approximately 9.6-times. In addition, the cleavage of caspase-3, -8, and -9 in cultured AGS cells treated with 5HHMF was significantly confirmed. Therefore, our results suggest that 5HHMF from H. fusiforme is involved in the induction of death receptorassociated apoptosis mediators in human gastric AGS carcinoma cells.
Journal of Microbiology and Biotechnology 12/2012; 22(12):1665-1672. · 1.40 Impact Factor
[show abstract][hide abstract] ABSTRACT: TGF-β-induced epithelial-mesenchymal transition (EMT) induces the proliferation and migration of the human lens epithelial (HLE) cells. Ganglioside GM3, a simple sialic acid-containing glycosphingolipids on mammalian cell membranes, regulates various pathological phenomena such as insulin resistance and tumor progression. However, the relation between ganglioside GM3 and TGF-β-induced EMT in the HLE B-3 cells is poorly understood. Here, we demonstrated that ganglioside GM3 was involved in TGF-β1-induced EMT in HLE B-3 cells. Our results indicated that the expression of ganglioside GM3 and GM3 synthase mRNA were significantly increased in TGF-β1-induced HLE B-3 cells. Reporter gene analysis also demonstrated that transcriptional activation of GM3 synthase gene was regulated by Sp1 in HLE B-3 cells upon TGF-β1 stimulation. Interestingly, the inhibition of ganglioside GM3 expression by d-PDMP and GM3 synthase shRNA resulted significantly in the suppression of cell migration and EMT-related signaling in HLE B-3 cells stimulated by TGF-β1. Furthermore, exogenous treatment of ganglioside GM3 rescued the expression of EMT-molecules and cell migration suppressed by the depletion of ganglioside GM3 in TGF-β1-induced HLE B-3 cells. We also found that ganglioside GM3 interacted with TGFβRs in TGF-β1-induced HLE B-3 cells. Taken together, these results suggest that ganglioside GM3 induced by TGF-β1 regulates the EMT by potential interaction with TGFβRs.
[show abstract][hide abstract] ABSTRACT: Cordycepin was purified from a mushroom, Cordyceps militaris, and its effect on Th1 and Th2 cytokines was examined. The level of cytokine induction in mouse splenocytes was estimated after co-inoculation of purified cordycepin and LPS. When 5 microg/ml of purified cordycepin was exposed to mouse splenocytes for 72 h, the level of a Th1 cytokine IL-12 increased by 2.9-fold. The addition of the purified cordycepin to splenocytes also increased the level of Th2 cytokines, IL-4 and IL-10, by 1.9- and 1.8- fold, respectively. Therefore, cordycepin increases the cytokine levels and may contribute to the up-regulation of cellular and humoral immunity.
Journal of Microbiology and Biotechnology 08/2012; 22(8):1161-4. · 1.40 Impact Factor
[show abstract][hide abstract] ABSTRACT: Apoptosis, the main type of programmed cell death, plays an essential role in a variety of biological events. Whereas "classical" apoptosis is dependent on caspase activation, caspase-independent death is increasingly recognized as an alternative pathway. To develop new anticancer agents, oleifolioside A was isolated from Dendropanax morbifera Leveille and the biochemical mechanisms of oleifolioside A-induced apoptosis in HeLa cells were investigated. Exposure to oleifolioside A resulted in caspase activation and typical features of apoptosis, although cell death was not prevented by caspase inhibition. Oleifolioside A treatment induced up-regulation of Bad, loss of mitochondrial membrane potential, nuclear relocation of mitochondrial factors, apoptosis-inducing factor (AIF), endonuclease G (EndoG), and apoptosis induction. This is the first report of anticancer activity of oleifolioside A, and nuclear translocation of AIF and EndoG in oleifolioside A-treated HeLa cells might represent an alternative death signaling pathway in the absence of caspase activity.
Journal of Agricultural and Food Chemistry 05/2012; 60(21):5400-6. · 2.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: The expression of matrix metalloproteinase (MMPs)-9 is critical for cell migration and can lead to invasion and metastasis of cancer cells. In the present study, we examined the inhibitory effects of JNP3, a new compound which was isolated from traditional Chinese medicine, on cell invasion and MMP-9 activation in phorbol myristate acetate (PMA)-induced MCF-7 cells. Treatment with JNP3 significantly and selectively inhibited PMA-induced MMP-9 secretion, mRNA expression and protein levels, and these results led to reduction of cell invasion and migration in PMA-induced MCF-7 cells. The results of MMP-9 promoter assay and EMSA showed that JNP3 specifically inhibited PMA-induced MMP-9 gene expression by blocking NF-κB-dependent transcriptional activity. In addition, PMA-induced phosphorylation of ERK1/2 and JNK were suppressed by JNP3 treatment, whereas the phosphorylation of p38 MAPK was not affected by JNP3. These results suggest that JNP3 can be potential anti-cancer agents through specific inhibition of NF-κB-dependent MMP-9 gene expression.
Biochemical and Biophysical Research Communications 04/2012; 421(2):190-6. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: In this study, the effect of cordycepin (3'-deoxyadenosine), a major component of Cordyceps militaris, an ingredient of traditional Chinese medicine was investigated for the first time on apoptotsis in human neuroblastoma SK-N-BE(2)-C and melanoma SK-MEL-2 cells. Cordycepin significantly inhibited the proliferation of human neuroblastoma SK-N-BE(2)-C and human melanoma SK-MEL-2 cells with IC50 values of 120 microM and 80 microM, respectively. Cordycepin treatment at 120 microM and 80 microM, respectively, induced apoptosis in both cells and caused the increase of cell accumulation in a time-dependent manner at the apoptotic sub-G1 phase, as evidenced by the flow cytometry (FCM) and annexin V-fluorescein isothiocyanate (FITC) analyses. Western blot analysis revealed the induction of active caspase-3 and poly(ADP-ribose)polymerase (PARP) cleavage by cordycepin treatment. These results suggest that cordycepin is a potential candidate for cancer therapy of neuroblastoma and melanoma.
Indian journal of biochemistry & biophysics 04/2012; 49(2):86-91. · 1.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: Oleifolioside A, a new triterpenoid compound isolated from Dendropanax morbifera Leveille (D. morbifera), was shown in this study to have potent inhibitory effects on lipopolysaccharide (LPS-)stimulated nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production in RAW 264.7 macrophages. Consistent with these findings, oleifolioside A was further shown to suppress the expression of LPS-stimulated inducible nitric oxide synthase (iNOS) and cyclooxigenase-2 (COX-2) in a dose-dependent manner at both the protein and mRNA levels and to significantly inhibit the DNA-binding activity and transcriptional activity of NF-κB in response to LPS. These results were found to be associated with the inhibition of the degradation and phosphorylation of IκB-α and subsequent translocation of the NF-κB p65 subunit to the nucleus. Inhibition of NF-κB activation by oleifolioside A was also shown to be mediated through the prevention of p38 MAPK and ERK1/2 phosphorylation. Taken together, our results suggest that oleifolioside A has the potential to be a novel anti-inflammatory agent capable of targeting both the NF-κB and MAPK signaling pathways.
Evidence-based Complementary and Alternative Medicine 01/2012; 2012:637512. · 1.72 Impact Factor
[show abstract][hide abstract] ABSTRACT: The present study evaluated the anticomplement effects from isolated compounds of black bean in classical pathway complement system. Using column chromatograph, four compounds kaempferol 3-O-β-D-glucopyranoside (1), kaempferol 5-O-β-D-glucopyranoside (2), 3-O-[α-L-rhamnopyranosyl(1-2)-β-D-glucopyranosyl(1-2)-β-D-glucuronopyranosyl]olean-12-en-3β,22β,24-triol (3) and 3-O-[β-D-glucopyranosyl(1-2)-β-D-galactopyranosyl(1-2)-β-D-glucuronopyranosyl]olean-12-en-3β,22β,24-triol (4) were isolated and evaluated for in vitro anticomplement activity. 3 showed inhibitory activity against complement system with 50% inhibitory concentrations (IC(50)) values of 98.2 μg/ml. This is the first report of anticomplement activity of isolated compounds from black bean.
Immunopharmacology and Immunotoxicology 11/2011; · 1.36 Impact Factor
[show abstract][hide abstract] ABSTRACT: In this study we investigated for the first time the transcriptional regulation of pig Galβ1,3GalNAc α2,3-sialyltransferase (pST3Gal I) in response to TGF-β1 in porcine kidney PK-15 cells. The pST3Gal I gene was found to span about 90kb and to be composed of 8 exons including 2 exons in the 5'-untranslated region. RT-PCR analysis indicated that the induction of pST3Gal I by TGF-β1 is regulated at the transcriptional level. Functional analysis of the 5'-flanking region of the pST3Gal I gene revealed the -1257 to -976 region functions as the TGF-β1-inducible promoter and that the Smad-binding site at -1020 is crucial for TGF-β1-induced expression of pST3Gal I in PK-15 cells. In addition, the transcriptional activity of pST3Gal I induced by TGF-β1 in PK-15 cells was strongly inhibited by SIS3, which is a specific Smad-3 inhibitor. In summary, our results identified the core promoter region in the pST3Gal I promoter and demonstrated that Smad-3 binding to the Samd-3 binding site at -1020 is essential for transcriptional activation of pST3Gal I in TGF-β1-induced PK-15 cells.
Biochemical and Biophysical Research Communications 09/2011; 414(1):159-64. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: The present study demonstrated that valproic acid (VPA) transcriptionally regulates human GM3 synthase (hST3Gal V), which catalyzes ganglioside GM3 biosynthesis in ARPE-19 human retinal pigment epithelial cells. For this, we characterized the promoter region of the hST3Gal V gene. Functional analysis of the 5'-flanking region of the hST3Gal V gene revealed that the -177 to -83 region functions as the VPA-inducible promoter and that the CREB/ATF binding site at -143 is crucial for VPA-induced expression of hST3Gal V in ARPE-19 cells. In addition, the transcriptional activity of hST3Gal V induced by VPA in ARPE-19 cells was inhibited by SP600125, a c-Jun N-terminal kinase (JNK) inhibitor. In summary, our results identified the core promoter region in the hST3Gal V promoter and for the first time demonstrated that ATF2 binding to the CREB/ATF binding site at -143 is essential for transcriptional activation of hST3Gal V in VPA-induced ARPE-19 cells.
[show abstract][hide abstract] ABSTRACT: We assessed the effects of chloroform extract of fermented Viola mandshurica (CEFV) on melanogenesis B16 melanoma cells. CEFV treatment significantly decreased melanin content and tyrosinase activity in dose-dependent manners. To elucidate the mechanism of the inhibitory effects of CEFV on melanogenesis, we performed RT-PCR and Western blotting for melanogenesis-related genes such as tyrosinase, tyrosinase-related protein-1 (TRP-1), TRP-2, and microphthalmia-associated transcription factor (MITF). CEFV strongly inhibited mRNA as well as the protein expression of tyrosinase and MITF, but had no significant effect on TRP-1 or TRP-2 expressions. It markedly decreased the phosphorylation of cAMP responsive element binding protein (CREB), and induced the duration of extracellular signal-regulated kinase (ERK) activation, leading to reduction of MITF expression and subsequently that of tyrosinase. Therefore, we suggest that CEFV induces downregulation of melanogenesis through decreased CREB phosphorylation and ERK activation.
Bioscience Biotechnology and Biochemistry 05/2011; 75(5):841-7. · 1.27 Impact Factor
[show abstract][hide abstract] ABSTRACT: The butanol-soluble fraction of the dried root of Angelica gigas exhibited significant protection against chloroquine-sensitive strains of Plasmodium falciparum using the parasite lactate dehydrogenase assay method. Using antiplasmodial activity-guided fractionation, five coumarins, marmesinin (1), nodakenin (2), skimmin (3), apiosylskimmin (4), and magnolioside (5), were isolated and evaluated for in vitro antiplasmodial activity, as well as for their cytotoxic potential on SK-OV-3 cancer cell lines. Compounds 1 and 5 showed notable growth inhibitory activity against chloroquine-sensitive strains of P. falciparum with IC(50) values of 5.3 and 8.2 μM. The compounds showed no significant cytotoxicity (IC(50) > 100 μM) toward the SK-OV-3 cancer cell line. This is the first report on the antiplasmodial activity of these coumarin derivatives from the dried root of A. gigas.
Immunopharmacology and Immunotoxicology 03/2011; 33(4):663-6. · 1.36 Impact Factor
[show abstract][hide abstract] ABSTRACT: Our previous study has demonstrated that the methanol extract of Hyul-Tong-Ryung (HM) specifically suppresses the phorbol 12-myristate 13-acetate (PMA)-induced matrix metalloproteinase-9 (MMP-9) production through the inhibition of MMP-9 mRNA expression in MCF-7 human breast carcinoma cells. However, the molecular mechanisms involved in transcriptional suppression of MMP-9 by HM in PMA-induced MCF-7 cells are not known. In this study, we aimed to elucidate the molecular mechanisms involved in the inhibition of MMP-9 expression by HM in PMA-induced MCF-7 cells. The results of promoter assay and EMSA showed that HM specifically inhibits MMP-9 gene expression by blocking PMA-stimulated activation of activator protein-1 (AP-1). In addition, PMA-stimulated phosphorylation of extracellular signal regulated kinase 1/2 (ERK 1/2) was suppressed by HM treatment, whereas the phosphorylation of either c-Jun N-terminal kinase (JNK) or p38 mitogen-activated protein kinase (MAPK) was not affected. HM could inhibit the PMA-induced MMP-9 expression through suppression of the transcriptional activity of MMP-9 gene in MCF-7 cells. These results indicate that HM inhibits PMA-induced MMP-9 expression by blocking the activation of activator protein-1 (AP-1) via extracellular signal regulated kinase 1/2 (ERK 1/2) signaling pathway.
Immunopharmacology and Immunotoxicology 12/2010; 32(4):600-6. · 1.36 Impact Factor
[show abstract][hide abstract] ABSTRACT: In this study, we have shown that gene expression of human GD3 synthase (hST8Sia I) is suppressed by triptolide (TPL) in human melanoma SK-MEL-2 cells. To elucidate the mechanism underlying the downregulation of hST8Sia I gene expression in TPL-treated SK-MEL-2 cells, we characterized the TPL-inducible promoter region within the hST8Sia I gene using luciferase constructs carrying 5'-deletions of the hST8Sia I promoter. Functional analysis of the 5'-flanking region of the hST8Sia I gene demonstrated that the -1146 to -646 region, which contains putative binding sites for transcription factors c-Ets-1, CREB, AP-1 and NF-κB, functions as the TPL-inducible promoter of hST8Sia I in SK-MEL-2 cells. Site-directed mutagenesis and ChIP analysis indicated that the NF-κB binding site at -731 to -722 is crucial for TPL-induced suppression of hST8Sia I in SK-MEL-2 cells. This suggests that TPL induces down-regulation of hST8Sia I gene expression through NF-κB activation in human melanoma cells.
Experimental and Molecular Medicine 11/2010; 42(12):849-55. · 2.57 Impact Factor
[show abstract][hide abstract] ABSTRACT: To determine which amino acid residues are essential for the catalytic activity of mouse Galbeta1,3GalNAc alpha2,3-sialyltransferase (mST3Gal I), chemical modification and site-directed mutagenesis were employed against tryptophan and cysteine residues located in the predicted catalytic domain. This enzyme was strongly inhibited by N-bromosuccinimide, a specific blocking reagent for tryptophan residues, and the enzyme activity was completely lost at 3 mM, suggesting the involvement of tryptophan residues in the catalytic activity of mST3Gal I. The N-ethylmaleimide, an irreversible reagent for sulfhydryl group, significantly inhibited the enzyme activity. Seven tryptophan and six cysteine residues conserved in the cloned Galbeta1,3GalNAc alpha2,3-sialyltransferases were separately substituted into phenylalanine and serine, respectively. The enzymatic activity assay for tryptophan mutants produced in COS cells showed a complete abolishment of the activity in all of the mutants, except that W70F and W97F retained about 60% and 40% activities of wild type, respectively. In the case of cysteine mutants, no enzyme activity was observed like tryptophan mutants, except for C139S. These results suggest that tryptophan and cysteine residues conserved in ST3Gal I are critical for its activity.
Indian journal of biochemistry & biophysics 06/2010; 47(3):135-40. · 1.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: Investigation of collagenase and gelatinase inhibitory natural components afforded two isoflavonoids. Two isoflavonoids, tectorigenin-7-O-β-D-glucoside (1) and luteolin-7-O-β-D-glucuronopyranoside (2), were isolated from ethyl acetate fraction of Viola patrinii fermentation extracts (VPFE). Of these, compounds 1 and 2 exhibited collagenase inhibitory activity (IC(50)) at a concentration of less than 1.5 μM, and compound 2 showed gelatinases A and B inhibitory activity (IC(50)) at 0.3 μM and 0.8 μM, respectively.
Immunopharmacology and Immunotoxicology 03/2010; 32(4):614-6. · 1.36 Impact Factor
[show abstract][hide abstract] ABSTRACT: A strain WL-11 with high laccase activity was isolated from activated sludge collected from the effluent treatment plant of a textile and dyeing industry. It was identified as Aeromonas hydrophila by physiological test and 16S rDNA sequence analysis. A gene encoding of laccase from a newly isolated Aeromonas hydrophila WL-11 was cloned and characterized. Nucleotide sequence analysis showed an open reading frame of 1605 bp encoding a polypeptide comprised of 534 amino acids. The primary structure of the enzyme predicted the structural features characteristic of other laccases, including the conserved regions of four histidine-rich copper-binding sites. The predicted amino acid sequence showed a high homology (more than 60%) with bacterial laccases in the genome and protein databases and the highest degree of similarity (61% identity) was observed with the multicopper oxidase of Klebsiella sp. 601. When expressed in Escherichia coli, the recombinant enzyme was overproduced in the cytoplasm as soluble and active form. The purified enzyme had an optimum pH of 2.6 and 8.0 for ABTS (2,2'-azino-bis(3-ethylbenzthiazolinesulfonic acid) and DMP (2,6-dimethoxyphenol), respectively. The kinetic study on ABTS revealed a higher affinity of this enzyme to this substrate than DMP.
Journal of Environmental Sciences 01/2010; 22(4):635-40. · 1.77 Impact Factor