Wahn Soo Choi

Suppression of inducible nitric oxide synthase and cyclooxygenase-2 expression in RAW 264.7 macrophages by sesquiterpene lactones.

The molecular mechanism underlying the suppression of lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma)-induced nitric oxide (NO) and prostaglandin (PG) E(2) production was investigated in RAW 264.7 macrophages treated with sesquiterpene lactones, zaluzanin-C and estafiatone, isolated from Ainsliaea. Zaluzanin-C and estafiatone decreased NO production in LPS/IFN-gamma-stimulated RAW 264.7 macrophages with an IC50 of about 6.61 microM and 3.80 microM, respectively. In addition, these compounds inhibited the synthesis of PGE(2) in LPS/IFN-gamma-treated RAW 264.7 macrophages. Furthermore, treatment with zaluzanin-C and estafiatone resulted in a decrease in inducible No Synthase (iNOS) and Cyclooxygenase-2 (COX-2) protein and mRNA expression levels. Zaluzanin-C and estafiatone inhibited nuclear factor-kappaB (NF-kappaB) activation, a transcription factor necessary for iNOS and COX-2 expression in response to LPS/IFN-gamma. This effect was accompanied by parallel reduction of phosphorylation and degradation of inhibitor of kappaB (IkB). In addition, these effects were completely blocked by treatment with cysteine, indicating that the inhibitory effect of zaluzanin-C and estafiatone might be mediated by alkylation of either NF-kappaB itself or an upstream molecule of NF-kappaB. These results demonstrate that the suppression of NF-kappaB activation by zaluzanin-C and estafiatone might be attributed to inhibition of nuclear translocation of NF-kappaB resulting from blockade of the degradation of IkappaB, leading to suppression of the expression of iNOS and COX-2, which play important roles in inflammatory signaling pathways.

Phospholipases D1 and D2 Regulate Different Phases of Exocytosis in Mast Cells

The rat mast cell line RBL-2H3 contains both phospholipase D (PLD)1 and PLD2. Previous studies with this cell line indicated that expressed PLD1 and PLD2 are both strongly activated by stimulants of secretion. We now show by use of PLDs tagged with enhanced green fluorescent protein that PLD1, which is largely associated with secretory granules, redistributes to the plasma membrane in stimulated cells by processes reminiscent of exocytosis and fusion of granules with the plasma membrane. These processes and secretion of granules are suppressed by expression of a catalytically inactive mutant of PLD1 or by the presence of 50 mM 1-butanol but not tert-butanol, an indication that these events are dependent on the catalytic activity of PLD1. Of note, cholera toxin induces translocation of PLD1-labeled granules to the plasma membrane but not fusion of granules with plasma membrane or secretion. Subsequent stimulation of calcium influx with Ag or thapsigargin leads to rapid redistribution of PLD1 to the plasma membrane and accelerated secretion. Also of note, PLD1 is recycled from plasma membrane back to granules within 4 h of stimulation. PLD2, in contrast, is largely confined to the plasma membrane, but it too participates in the secretory process, because expression of catalytically inactive PLD2 also blocks secretion. These data indicate a two-step process: translocation of granules to the cell periphery, regulated by granule-associated PLD1, and a calcium-dependent fusion of granules with the plasma membrane, regulated by plasma membrane-associated PLD2 and possibly PLD1.

Norepinephrine induces VEGF expression and angiogenesis by a hypoxia-inducible factor-1α protein-dependent mechanism.

A growing number of studies have demonstrated that physiological factors can influence the progression of several cancers via cellular immune function, angiogenesis and metastasis. Recently, stress‐induced catecholamines have been shown to increase the expression of various cancer progressive factors, including vascular endothelial growth factor (VEGF), matrix metalloproteinases and interleukins. However, a detailed mechanism remains to be identified. In this study, we investigated the role of adrenergic receptors and hypoxia‐inducible factor (HIF)‐1α protein in catecholamine‐induced VEGF expression and angiogenesis. Treatment of the cells with norepinephrine (NE) or isoproterenol induced VEGF expression and HIF‐1α protein amount in a dose‐dependent manner. Induction of VEGF expression by NE was abrogated when the cells were transfected with HIF‐1α–specific siRNA. Similarly, adenylate cyclase activator forskolin and cyclic AMP‐dependent protein kinase A inhibitor H‐89 enhanced and decreased HIF‐1α protein amount, respectively. More importantly, conditioned medium of NE‐stimulated cancer cells induced angiogenesis in a HIF‐1α protein–dependent manner. In addition, pretreatment of cells with propranolol, a β‐adrenergic receptor (AR) blocker, completely abolished induction of VEGF expression and HIF‐1α protein amount by NE in all of the tested cancer cells. However, treatment with the α1‐AR blocker prazosin inhibited NE‐induced HIF‐1α protein amount and angiogenesis in SK‐Hep1 and PC‐3 but not MDA‐MB‐231 cells. Collectively, our results suggest that ARs and HIF‐1α protein have critical roles in NE‐induced VEGF expression in cancer cells, leading to stimulation of angiogenesis. These findings will help to understand the mechanism of cancer progression by stress‐induced catecholamines and design therapeutic strategies for cancer angiogenesis.

Curcumin, a constituent of curry, suppresses IgE-mediated allergic response and mast cell activation at the level of Syk

BACKGROUND Activation of mast cells through the high-affinity receptor for IgE (FcepsilonRI) underlies atopic allergic reactions. Curcumin can block this activation, but the mechanism and the effects of curcumin on IgE-mediated allergic reactions are unknown. OBJECTIVES We sought to determine the antiallergic activity of curcumin in vivo and its mechanism of action in mast cells. METHODS The antiallergic activity of curcumin was evaluated in mast cell cultures and the passive cutaneous anaphylaxis model. The effects of curcumin on mast cell signaling events were examined by using immunoblotting, immunoprecipitation, RT-PCR, and other molecular biologic approaches. RESULTS Curcumin inhibited antigen-mediated activation of mast cells and passive cutaneous anaphylaxis in mice. Suppression of degranulation and secretion of TNF-alpha and IL-4 was apparent at concentrations as low as 3 micromol/L curcumin in activated mast cells. Similar concentrations of curcumin suppressed Syk-dependent phosphorylations of the adaptor proteins linker of activated T cells and Grb2-associated binder 2, which are critical for mast cell activation. Although curcumin did not inhibit the phosphorylation of Syk itself, it directly inhibited Syk kinase activity in vitro. Further downstream, activating phosphorylations of Akt and the mitogen-activated protein kinases p38, p44/42 (extracellular signal-regulated kinase 1/2), and c-Jun N-terminal kinase, which are critical for the production of inflammatory cytokines, were also inhibited. CONCLUSIONS Curcumin inhibits Syk kinase-dependent signaling events in mast cells and might thus contribute to its antiallergic activity. Therefore curcumin might be useful for the treatment of mast cell-related immediate and delayed allergic diseases.

Tumor necrosis factor α–induced interleukin-32 is positively regulated via the Syk/protein kinase Cδ/JNK pathway in rheumatoid synovial fibroblasts

OBJECTIVE Interleukin-32 (IL-32) is a recently discovered cytokine that appears to play a critical role in human rheumatoid arthritis (RA). It is highly expressed in synovium and fibroblast-like synoviocytes (FLS) from RA patients, but not in patients with osteoarthritis (OA). This study was undertaken to assess IL-32 levels in RA synovial fluid (SF) and to investigate the secretion and regulation of IL-32 in RA FLS. METHODS FLS and SF were obtained from the joints of RA patients. The secretion and expression of IL-32 and activation of signaling molecules were examined by enzyme-linked immunosorbent assay, immunoblotting, immunoprecipitation, reverse transcriptase-polymerase chain reaction, and small interfering RNA (siRNA) transfection. RESULTS IL-32 levels were high in RA SF compared with OA SF. Furthermore, RA FLS expressed and secreted IL-32 when stimulated with tumor necrosis factor alpha (TNFalpha). TNFalpha-induced expression of IL-32 was significantly suppressed, in a dose-dependent manner, by inhibitors of Syk, protein kinase Cdelta (PKCdelta), and JNK and by knockdown of these kinases and c-Jun with siRNA. We also observed that PKCdelta mediated the activation of JNK and c-Jun, and experiments using specific inhibitors and siRNA demonstrated that Syk was the upstream kinase for the activation of PKCdelta. CONCLUSION The present findings suggest that IL-32 may be a newly identified prognostic biomarker in RA, thereby adding valuable knowledge to the understanding of this disease. The results also demonstrate that the production of IL-32 in RA FLS is regulated by Syk/PKCdelta-mediated signaling events.

Resveratrol enhances chemosensitivity of doxorubicin in multidrug-resistant human breast cancer cells via increased cellular influx of doxorubicin

BACKGROUND Multidrug resistance is a major problem in the treatment of breast cancer, and a number of studies have attempted to find an efficient strategy with which to overcome it. In this study, we investigate the synergistic anticancer effects of resveratrol (RSV) and doxorubicin (Dox) against human breast cancer cell lines. METHODS The synergistic effects of RSV on chemosensitivity were examined in Dox-resistant breast cancer (MCF-7/adr) and MDA-MB-231 cells. In vivo experiments were performed using a nude mouse xenograft model to investigate the combined sensitization effect of RSV and Dox. RESULTS AND CONCLUSION RSV markedly enhanced Dox-induced cytotoxicity in MCF-7/adr and MDA-MB-231 cells. Treatment with a combination of RSV and Dox significantly increased the cellular accumulation of Dox by down-regulating the expression levels of ATP-binding cassette (ABC) transporter genes, MDR1, and MRP1. Further in vivo experiments in the xenograft model revealed that treatment with a combination of RSV and Dox significantly inhibited tumor volume by 60%, relative to the control group. GENERAL SIGNIFICANCE These results suggest that treatment with a combination of RSV and Dox would be a helpful strategy for increasing the efficacy of Dox by promoting an intracellular accumulation of Dox and decreasing multi-drug resistance in human breast cancer cells.

Protective effects of Gastrodia elata Blume on MPP+-induced cytotoxicity in human dopaminergic SH-SY5Y cells.

AIM OF THE STUDY Gastrodia elata (GE) Blume (Orchidaceae) has been traditionally used as a folk medicine in Oriental countries since centuries for their variety of therapeutic benefits. This study is an attempt to investigate the protective effects of GE extract against MPP(+)-induced cytotoxicity in human dopaminergic SH-SY5Y cells and explore the neuroprotective mechanisms involved. MATERIALS AND METHODS Human dopaminergic SH-SY5Y cells were used to demonstrate the protective effects of GE against multiple parameters such as MPP(+)-induced cell viability, oxidative damage, expression of Bcl-2 and Bax, caspase-3 and poly(ADP-ribose) polymerase proteolysis. RESULTS GE effectively attenuated the cytotoxicity and improved cell viability in a dose-dependent manner. GE was effective in inhibiting both, the increased production of reactive oxygen species (ROS) and increase in Bax/Bcl-2 ratio, cleaved caspase-3 and PARP proteolysis. CONCLUSION Data from this study suggests the protective effects of GE on MPP(+)-induced cytotoxicity in dopaminergic cells, which may be ascribed to its significant anti-oxidative and anti-apoptotic properties, thus, GE might prove to be a valuable therapeutic agent for the treatment of various neurodegenerative diseases including progressive Parkinsons disease (PD).

Gene Transfer of Redox Factor-1 Inhibits Neointimal Formation: Involvement of Platelet-Derived Growth Factor-β Receptor Signaling via the Inhibition of the Reactive Oxygen Species–Mediated Syk Pathway

The role of apurinic/apyrimidinic endonuclease-1/redox factor-1 (Ref-1) in vascular smooth muscle cells has yet to be clearly elucidated. Therefore, we attempted to determine the roles of Ref-1 in the migration induced by platelet-derived growth factor (PDGF)-BB and in its signaling in rat aortic smooth muscle cells (RASMCs). Cellular migration, superoxide (O2−·) production, Rac-1 activity, and neointima formation were determined in cells transfected with adenoviruses encoding for Ref-1 (AdRef-1) and small interference RNA of Ref-1. Overexpression of Ref-1 induced by treatment with RASMCs coupled with AdRef-1 inhibited the migration induced by PDGF-BB. PDGF-BB also increased the phosphorylation of the PDGF&bgr; receptor, spleen tyrosine kinase (Syk), mitogen-activated protein kinase, and heat shock protein 27, but these increases were significantly inhibited by AdRef-1 treatment. PDGF-BB increased O2−· production and Rac-1 activity, and these were diminished in cells transfected with AdRef-1. In contrast, RASMC migration, phosphorylation of Syk and O2−· production in response to PDGF-BB were increased by the knock down of Ref-1 with small interference RNA. The phosphorylation of PDGF&bgr; receptor in response to PDGF-BB was inhibited completely by the Syk inhibitor and was partly attenuated by a NADPH oxidase inhibitor. PDGF-BB increased the sprout outgrowth of the aortic ring ex vivo, which was inhibited in the AdRef-1–infected RASMCs as compared with the controls. Balloon injury–induced neointimal formation was significantly attenuated by the gene transfer of AdRef-1. These results indicate that Ref-1 inhibits the PDGF-mediated migration signal via the inhibition of reactive oxygen species–mediated Syk activity in RASMCs.

Sirtinol, a class III HDAC inhibitor, induces apoptotic and autophagic cell death in MCF-7 human breast cancer cells

Sirtuins (SIRTs), NAD+-dependent class III histone deacetylases (HDACs), play an important role in the regulation of cell division, survival and senescence. Although a number of effective SIRT inhibitors have been developed, little is known about the specific mechanisms of their anticancer activity. In this study, we investigated the anticancer effects of sirtinol, a SIRT inhibitor, on MCF-7 human breast cancer cells. Apoptotic and autophagic cell death were measured. Sirtinol significantly inhibited the proliferation of MCF-7 cells in a concentration-dependent manner. The IC50 values of sirtinol were 48.6 µM (24 h) and 43.5 µM (48 h) in MCF-7 cells. As expected, sirtinol significantly increased the acetylation of p53, which has been reported to be a target of SIRT1/2. Flow cyto-metry analysis revealed that sirtinol significantly increased the G1 phase of the cell cycle. The upregulation of Bax, downregulation of Bcl-2 and cytochrome c release into the cytoplasm, which are considered as mechanisms of apoptotic cell death, were observed in the MCF-7 cells treated with sirtinol. The annexin V-FITC assay was used to confirm sirtinol-induced apoptotic cell death. Furthermore, the expression of LC3-II, an autophagy-related molecule, was significantly increased in MCF-7 cells after sirtinol treatment. Autophagic cell death was confirmed by acridine orange and monodansylcadaverine (MDC) staining. Of note, pre-treatment with 3-methyladenine (3-MA) increased the sirtinol-induced MCF-7 cell cytotoxicity, which is associated with blocking autophagic cell death and increasing apoptotic cell death. Based on our results, the downregulation of SIRT1/2 expression may play an important role in the regulation of breast cancer cell death; thus, SIRT1/2 may be a novel molecular target for cancer therapy and these findings may provide a molecular basis for targeting SIRT1/2 in future cancer therapy.

Interleukin-33 stimulates formation of functional osteoclasts from human CD14(+) monocytes.

Interleukin (IL)-33 is a recently described pro-inflammatory cytokine. Here we demonstrate IL-33 as a regulator of functional osteoclasts (OCs) from human CD14+ monocytes. IL-33 stimulates formation of tartrate-resistant acid phosphatase (TRAP)+ multinuclear OCs from monocytes. This action was suppressed by anti-ST2 antibody, suggesting that IL-33 acts through its receptor ST2, but not by the receptor activator of NF-κB ligand (RANKL) decoy, osteoprotegerin, or anti-RANKL antibody. IL-33 stimulated activating phosphorylations of signaling molecules in monocytes that are critical for OC development. These included Syk, phospholipase Cγ2, Gab2, MAP kinases, TAK-1, and NF-κB. IL-33 also enhanced expression of OC differentiation factors including TNF-α receptor-associated factor 6 (TRAF6), nuclear factor of activated T cells cytoplasmic 1, c-Fos, c-Src, cathepsin K, and calcitonin receptor. IL-33 eventually induced bone resorption. This study suggests that the osteoclastogenic property of IL-33 is mediated through TRAF6 as well as the immunoreceptor tyrosine-based activation motif-dependent Syk/PLCγ pathway in human CD14+ monocytes.