Chang-Duk Jun

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.

Synergistic cooperation between water-soluble chitosan oligomers and interferon-γ 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.

Role of intracellular calcium as a priming signal for the induction of nitric oxide synthesis in murine peritoneal macrophages

Because the role of intracellular Ca2+ in the two‐signal process for the induction of nitric oxide (NO) synthesis is controversial, this study was undertaken to examine the role of Ca2+ in the transcriptional regulation of inducible NO synthase (iNOS) in murine peritoneal macrophages. Treatment of the cells with thapsigargin (TG) or 2,5‐di‐(t‐butyl)‐1,4‐benzodihydroquinone (tBuBHQ), which are the specific and potent Ca2+‐ATPase inhibitors of endoplasmic reticulum (ER), showed modest effects on tumoricidal function, whereas TG or tBuBHQ in combination with interferon‐γ (IFN‐γ) or lipopolysaccharide (LPS) showed marked effects on tumoricidal function of the cells. The tumoricidal effects of the activated macrophages were correlated with the amount of NO synthesis, and totally abrogated by the use of NOS inhibitor, N G‐monomethyl‐l‐arginine (NGMMA). The increases in NO synthesis was reflected as increased amounts of iNOS mRNA by Northern blotting. To confirm that iNOS induction was due to the changes in the intracellular Ca2+ level, the acetoxymethyl ester of 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA‐AM), an intracellular Ca2+ chelator, was used. Blocking the increase of cytosolic free Ca2+ significantly decreased the induction of NO synthesis. To demonstrate that intracellular Ca2+ acts as a ‘priming’ signal rather than a ‘triggering’ signal on the induction of NO synthesis by murine peritoneal macrophages, we designed several experiments. When the cells were treated with TG 6 hr after the treatment with IFN‐γ, there was no increase in NO synthesis. In addition, when the cells were treated with TG or LPS 6 hr after treatment with tBuBHQ, a synergistic increase on NO synthesis was shown only in the case of LPS. When phorbol 12‐myristate 13‐acetate (PMA), a protein kinase C (PKC) activator, was added to the cells 6 hr after the treatment with TG, there was a marked co‐operative induction of NO synthesis, even though PMA alone has no effect. Based on the results obtained in this study, we suggest that cytosolic Ca2+ might be enough for the expression of iNOS gene as a priming signal and PKC might be involved in the induction of NO synthesis as a triggering signal by post‐transcriptional modification of iNOS mRNA or iNOS itself in the activated murine peritoneal macrophages.

Inhibition of nitric oxide synthesis by butanol fraction of the methanol extract of Ulmus davidiana in murine macrophages

Since there is increasing evidence that nitric oxide (NO) plays a crucial role in the pathogenesis of inflammatory diseases, this study was undertaken to address whether the methanol (MeOH) extract and its fractions of the bark of Ulmus davidiana Planch (Ulmaceae) could modulate the expression of inducible NO synthase (iNOS) in thioglycollate-elicited murine peritoneal macrophages and murine macrophage cell line, RAW264.7 cells. Stimulation of the peritoneal macrophages and RAW264.7 cells with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) resulted in increased production of NO in the medium. However, the butanol (BuOH) fraction of the MeOH extract of U. davidiana barks showed marked inhibition of NO synthesis in a dose-dependent manner. The inhibition of NO synthesis was reflected in the decreased amount of iNOS protein, as determined by Western blotting. The BuOH fraction did not affect the viability of RAW264.7 cells, as assessed by methylthiazol-2-yl-2, 5-diphenyl tetrazolium bromide (MTT) assay; rather, it reduced endogenous NO-induced apoptotic cell death via inhibition of NO synthesis in RAW264.7 cells. On the other hand, the BuOH fraction showed no inhibitory effect on the synthesis of NO by RAW264.7 cells, when iNOS was already expressed by the stimulation with IFN-gamma and LPS. Collectively, these results demonstrate that the BuOH fraction inhibits NO synthesis by inhibition of the induction of iNOS in murine macrophages.

An efficient method for the rapid establishment of Epstein‐Barr virus immortalization of human B lymphocytes

Abstract. Several methods have been developed for the immortalization of B lymphocytes by Epstein‐Barr virus (EBV). We developed an efficient method which reduces the time from culture initiation to immortalization and cryopreservation. Two infections of EBV to lymphocytes, and the use of phorbol ester‐induced EBV stock significantly improved immortalization efficiency and reduced the time between initiation and immortalization and cryopreservation. The resulting cell bank was used to produce DNA for genetic studies focusing on the genes involved in immune and autistic disorders.

p38 kinase and c-jun N-terminal kinase oppositely regulates tumor necrosis factor α-induced vascular cell adhesion molecule-1 expression and cell adhesion in chondrosarcoma cells

We investigated signaling pathways leading to tumor necrosis factor (TNF) α‐induced intercellular adhesion molecule (ICAM)‐1 and vascular cell adhesion molecule (VCAM)‐1 expression in chondrosarcoma cells, and determined the functional significance of their expression by examining Jurkat T cell adhesion. TNF αinduced VCAM‐1 and ICAM‐1 expression and Jurkat T cell binding. Antibody blocking assay indicated that VCAM‐1 mediates TNF α‐induced Jurkat T cell adhesion. TNF αcaused activation of mitogen‐activated protein (MAP) kinase subtypes, extracellular signal‐regulated protein kinase, p38 kinase, and c‐jun N‐terminal kinase (JNK). ICAM‐1 expression was not altered by the inhibition of MAP kinases. However, VCAM‐1 expression and Jurkat T cell adhesion was blocked by the inhibition of p38 kinase, whereas inhibition of JNK enhanced VCAM‐1 expression and cell adhesion without any modulation of NF κB activation. Our results, therefore, indicate that p38 kinase mediates TNF α‐induced VCAM‐1 expression and cell adhesion, whereas JNK suppresses VCAM‐1 expression that is independent to NF κB activation.

Cyclic Adenosine Monophosphate Inhibits Ursolic Acid-Induced Apoptosis via Activation of Protein Kinase A in Human Leukaemic HL-60 Cells

This study was designed to investigate the effect of cAMP on ursolic acid-induced apoptosis of HL-60 cells. Ursolic acid decreased the viability of the cells in a dose-dependent manner, which was revealed as an apototic process characterized by ladder-pattern DNA fragmentation in agarose gel electrophoresis and segmented nuclei in DAPI-sulpharhodamin 101 staining. Ursolic acid-induced apoptosis of the cells was markedly inhibited by the addition of cAMP-elevating agents including DB-cAMP, CPT-cAMP, 8-Br-cAMP and forskolin. These results were further evidenced by the fact that inhibitors of cAMP-dependent protein kinase including H89 and KT5720 completely inhibited the cAMP-mediated rescue of HL-60 cells from ursolic acid-induced apoptosis. In addition, differentiating agents of the cells such as dimethyl sulfoxide and retinoic acid did not affect the ursolic acid-induced apoptosis of HL-60 cells. These results suggest that signaling pathway of cAMP-dependent activation of protein kinase A may affect the responsiveness of tumor cells upon ursolic acid.

Transcriptional regulation of IL‐8 by iron chelator in human epithelial cells is independent from NF‐κB but involves ERK1/2‐ and p38 kinase‐dependent activation of AP‐1

We have shown that the bacterial iron chelator, deferoxamine (DFO), triggers inflammatory signals including the production of CXC chemokine IL‐8, in human intestinal epithelial cells (IECs) by activating the ERK1/2 and p38 kinase pathways. In this study we investigated the mechanisms involved in IL‐8 generation by DFO, focusing on the transcription factors involved and the roles of both mitogen‐activated protein kinases (MAPKs) in the transcription factor activation. Treatment of human epithelial HT‐29 cells with DFO markedly up‐regulated the expression of the essential components of the transcription factor AP‐1 at a transcriptional level, while it minimally affected the expression of the NF‐κB subunits. DFO also induced AP‐1‐dependent transcriptional activity in HT‐29 cells, and this activity was further augmented by the wild‐type c‐Jun transfection. In contrast, the AP‐1 activity by DFO was markedly decreased by the dominant‐negative c‐Jun transfection. Electrophoretic mobility shift assays revealed that DFO increases the specific binding of AP‐1 but not of NF‐κB. Such AP‐1 binding and transcriptional activities were blocked by the inhibitors of the ERK1/2 and p38 kinase pathways, suggesting that both mitogen‐activated protein kinases (MAPKs) lie upstream of AP‐1. Besides its action on AP‐1, DFO also induced the specific binding of other transcription factors such as CREB and Egr‐1. In summary, our results indicate that iron chelator‐induced IL‐8 generation in IECs involves activation of ERK1/2 and p38 kinase and downstream activation of AP‐1. A possible link between iron status and two additional transcription factors, that is, CREB and Egr‐1, rather than NF‐κB, was also suggested. J. Cell. Biochem. 102: 1442–1457, 2007.

Alkamides from the fruits of Piper longum and Piper nigrum displaying potent cell adhesion inhibition.

Eight alkamides 1-8 were isolated by bioassay-guided isolation of EtOH extracts of the fruits of Piper longum and Piper nigum (Piperaceae). Their structures were elucidated by spectroscopic analysis ((1)H, (13)C NMR, and ESI-MS) as follows: guineensine (1), retrofracamide C (2), (2E,4Z,8E)-N-[9-(3,4-methylenedioxyphenyl)-2,4,8-nonatrienoyl]piperidine (3), pipernonaline (4), piperrolein B (5), piperchabamide D (6), pellitorin (7), and dehydropipernonaline (8). Their compounds 3-5, 7, and 8 inhibited potently the direct binding between sICAM-1 and LFA-1 of THP-1 cells in a dose-dependent manner, with IC(50) values of 10.7, 8.8, 13.4, 13.5, and 6.0 microg/mL, respectively.

Catalposide, a compound isolated from Catalpa Ovata, attenuates induction of intestinal epithelial proinflammatory gene expression and reduces the severity of trinitrobenzene sulfonic acid-induced colitis in mice

Certain irinoid-producing plants have been used as herbal anti-inflammatory remedies. Here we evaluated whether catalposide (CATP), a single compound isolated from irinoid-producing plant Catalpa ovata, has a potential for preventing or ameliorating diseases characterized by mucosal inflammation. Preliminary microarray-based gene expression test revealed that CATP, which alone did not significantly affect expression of any of the >8,000 genes analyzed, attenuated the expression of tumor necrosis factor-alpha (TNF-alpha)-induced proinflammatory genes including interleukin-8 (IL-8) in human intestinal epithelial HT-29 cells. Down-regulation of IL-8 mRNA accumulation was also reflected by the decreased IL-8 secretion in CATP-treated HT-29 cells. The signal transduction study revealed that CATP significantly attenuates TNF-alpha-mediated p38 and extracellular signal-regulated kinase (ERK) phosphorylation. Further, CATP reduced NF-kappaB-mediated transcriptional activation as well as Ikappa-Balpha degradation. To establish the in vivo relevance of these findings, we examined whether CATP could affect intestinal inflammation in vivo using the mouse model of trinitrobenzene sulfonic acid (TNBS)-induced inflammatory colitis. Intrarectal administration of CATP dramatically reduced the weight loss, colonic damage, and mucosal ulceration that characterize TNBS colitis. Moreover, CATP suppressed the expression of TNF-alpha, interleukin-1beta, and intercellular adhesion molecule-1 along with the inhibition of NF-kappa B p65 translocation into nucleus in TNBS colitis. Collectively, current results demonstrate that CATP may be an effective agent for the treatment of diseases characterized by mucosal inflammation.