Human ST3Gal II and ST6GalNAc IV genes increase human serum-mediated cytotoxicity to xenogeneic cells.

Carbohydrate-antigens widely existed on glycoproteins and glycosphingolipids of all mammalian cells play a crucial role in self-defense and immunity. Xeno-reactive antibodies included in natural human sera play a protecting role in an acute phase-rejection of xenotransplantation. In this study, we investigated the effect of an alteration of glycosylation-pattern, caused by human sialyltransferases such as hST3Gal II or hST6GalNAc IV, on human serum mediated cytotoxicity in pig kidney PK15 cells. From LDH cytotoxicity assay, cytotoxicity to human serum was significantly increased in hST3Gal II and hST6GalNAc IV-transfected PK15 cells, as compared to the control. In the hST6Gal I-carrying cells, the cytotoxicity to human serum was rather decreased. Moreover, flow cytometry analysis revealed that an alteration of pig glycosylation-pattern by hST3Gal II or hST6GalNAc IV influences on a binding of human IgM or IgG, respectively, in pig kidney cells, regardless of Gal antigen alteration. Finally, we found that hST6GalNAc IV contributed to increase of terminal disialylated tetrasaccharide structure, disialyl T antigen, as evidenced by increase of the MAL II lectin binding capacity in the hST6GalNAc IV-transfected PK15 cells, compared with control. Therefore, our results suggest that carbohydrate antigens, such as disialyl T antigen, newly synthesized by the ST3Gal II- and ST6GalNAc IV are potentially believed to be new xeno-reactive elements.

Up-regulation of inflammatory reactions by MPT32, a secreted protein of Mycobacterium tuberculosis in RAW264.7 macrophages.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb) and is still one of the global health burdens. The occurrence of various cases and multidrug resistance confirm that TB has not been completely conquered. For these reasons, the present research has been conducted to explore TB vaccine and drug candidate possibility using Mtb-secreted proteins. Among these proteins, MPT32 is known to have antigenicity and immunogenicity. There has not been a report on the host immune responses and regulation in macrophage cells. The present study was conducted with MPT32 in RAW 264.7 murine macrophage cells that control immune responses by sensing pathogen invasion and environmental change. We have found that MPT32 could activate lipopolysaccharide (LPS)-induced gene expression of metalloproteinase-9 (MMP-9) and inflammation in RAW 264.7 cells. After treating cells with MPT32, the increase in pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß (IL-1ß) and IL-6, was observed. In addition, activated macrophages expressed inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) to generate various inflammatory mediator molecules, such as nitric oxide (NO). The increase in iNOS and COX-2 levels, which are up-regulators of MMP-9 expression, was also confirmed. The biochemical events are involved in the downstream of activated MAPK signaling and translocation of NF-κ B transcription factor. The present results prove the immunomodulatory effect of MPT32 in the RAW 264.7 murine macrophage cells. it claims the possibility of a TB vaccination and drug candidate using MPT32, contributing to the prevention of TB.

T7 phage display reveals NOLC1 as a GM3 binding partner in human breast cancer MCF-7 cells.

Ganglioside GM3 is a simple monosialoganglioside (NeuAc-Gal-Glc-ceramide) that modulates cell adhesion, proliferation, and differentiation. Previously, we reported isolation of GM3-binding vascular endothelial growth factor receptor and transforming growth factor-ß receptor by the T7 phage display method (Chung et al., 2009; Kim et al., 2013). To further identify novel proteins interacting with GM3, we extended the T7 phage display method in this study. After T7 phage display biopanning combined with immobilized biotin-labeled 3'-sialyllactose prepared on a streptavidin-coated microplate, we isolated 100 candidate sequences from the human lung cDNA library. The most frequently detected clones from the blast analysis were the human nucleolar and coiled-body phosphoprotein 1 (NOLC1) sequences. We initially identified NOLC1 as a molecule that possibly binds to GM3 and confirmed this binding ability using the glutathione S-transferase fusion protein. Herein, we report another GM3-interacting protein, NOLC1, that can be isolated by the T7 phage display method. These results are expected to be helpful for elucidating the functional roles of ganglioside GM3 with NOLC1. When human breast cancer MCF-7 cells were examined for subcellular localization of NOLC1, immunofluorescence of NOLC1 was observed in the intracellular region. In addition, NOLC1 expression was increased in the nucleolus after treatment with the anticancer drug doxorubicin. GM3 and NOLC1 levels in the doxorubicin-treated MCF-7 cells were correlated, indicating possible associations between GM3 and NOLC1. Therefore, direct interactions between carbohydrates and cellular proteins can pave the path for new signaling phenomena in biology.

Regulatory mechanism for the human glioblastoma cell-specific expression of the human GD1c/GT1a/GQ1b synthase (hST8Sia V) gene.

In this study we observed that human GD1c/GT1a/GQ1b synthase (hST8Sia V) is particularly expressed in human glioblastoma cells. To address the mechanism regulating human glioblastoma-specific gene expression of the hST8Sia V, after the transcription start site (TSS) was identified by the 5'-rapid amplification of cDNA end with total RNA from human glioblastoma U87MG cells, the 5'-flanking region (2.5 kb) of the hST8Sia V gene was isolated and its promoter activity was examined. By luciferase reporter assay, this 5'-flanking region revealed strong promoter activity in only U-87MG cells, but not in other tissue-derived cancer cells. 5'-deletion mutant analysis showed that the region from -1140 to -494 is crucial for transcription of the hST8Sia V gene in U87MG cells. This region contains the activator protein-1 (AP-1) binding site, the main target of the c-Jun N-terminal kinase (JNK) downstream. The AP-1 binding site at -1043/-1037 was proved to be indispensable for the hST8Sia V gene-specific expression in U87MG cells by site-directed mutagenesis. Moreover, the transcriptional activation of hST8Sia V gene in U87MG cells was strongly inhibited by a specific JNK inhibitor, SP600125. These results suggest that the hST8Sia V gene-specific expression in U87MG cells is controlled by JNK/AP-1 signaling pathway.

Involvement of the p38 MAPK-NLRC4-Caspase-1 Pathway in Ionizing Radiation-Enhanced Macrophage IL-1ß Production.

Macrophages are abundant immune cells in the tumor microenvironment and are crucial in regulating tumor malignancy. We previously reported that ionizing radiation (IR) increases the production of interleukin (IL)-1ß in lipopolysaccharide (LPS)-treated macrophages, contributing to the malignancy of colorectal cancer cells; however, the mechanism remained unclear. Here, we show that IR increases the activity of cysteine-aspartate-specific protease 1 (caspase-1), which is regulated by the inflammasome, and cleaves premature IL-1ß to mature IL-1ß in RAW264.7 macrophages. Irradiated RAW264.7 cells showed increased expression of NLRC4 inflammasome, which controls the activity of caspase-1 and IL-1ß production. Silencing of NLRC4 using RNA interference inhibited the IR-induced increase in IL-1ß production. Activation of the inflammasome can be regulated by mitogen-activated protein kinase (MAPK)s in macrophages. In RAW264.7 cells, IR increased the phosphorylation of p38 MAPK but not extracellular signal-regulated kinase and c-Jun N-terminal kinase. Moreover, a selective inhibitor of p38 MAPK inhibited LPS-induced IL-1ß production and NLRC4 inflammasome expression in irradiated RAW264.7 macrophages. Our results indicate that IR-induced activation of the p38 MAPK-NLRC4-caspase-1 activation pathway in macrophages increases IL-1ß production in response to LPS.

Regulation of human ß-galactoside α2,6-sialyltransferase (hST6Gal I) gene expression during differentiation of human osteoblastic MG-63 cells.

In this study, we found that gene expression of the human ß-galactoside α2,6-sialyltransferase (hST6Gal I) was specifically increased during differentiation of human MG-63 osteoblastic cells by serum starvation (SS). In parallel, a distinct increase in binding to SNA, the α2,6-sialyl-specific lectin, was observed in serum-starved cells, as demonstrated by FACS analysis. 5'-Rapid amplification of cDNA ends analysis demonstrated that the increase of hST6Gal I transcript by SS is mediated by P1 promoter. To elucidate transcriptional regulation of hST6Gal I in SS-induced MG-63 cells, we functionally characterized the P1 promoter region of the hST6Gal I gene. The 5'-deletion analysis of P1 promoter region revealed that the 189 bp upstream region of transcription start site is critical for transcriptional activity of hST6Gal I gene in SS-induced MG-63 cells. This region contains the predicted binding sites for several transcription factors, including AREB6, FOXP1, SIX3, HNF1, YY2, and MOK2. The mutagenesis analysis for these sites and chromatin immunoprecipitation assay demonstrated that the YY2 binding site at -98 to -77 was essential for the SS-induced hST6Gal I gene expression during differentiation of MG-63 cells.

3'-sialyllactose targets cell surface protein, SIGLEC-3, and induces megakaryocyte differentiation and apoptosis by lipid raft-dependent endocytosis.

3'-sialyllactose is one of the abundant components in human milk oligosaccharides (HMOs) that protect infants from various viral infections in early stages of immune system development. 3SL is a combination of lactose and sialic acid. Most sialic acids are widely expressed in animal cells and they bind to siglec proteins. In this study, we demonstrate that 3SL specifically binds to CD33. It induces megakaryocyte differentiation and subsequent apoptosis by targeting cell surface protein siglec-3 (CD33) in human chronic myeloid leukemia K562 cells. The 3SL-bound CD33 was internalized to the cytosol via caveolae-dependent endocytosis. At the molecular level, 3SL-bound CD33 recruits the suppressor of cytokine signaling 3 (SOCS3) and SH2 domain-containing protein tyrosine phosphatase 1 (SHP1). SOCS3 is degraded with CD33 by proteasome degradation, while SHP-1 activates extracellular signal-regulated kinase (ERK) to induce megakaryocytic differentiation and subsequent apoptosis. The present study, therefore, suggests that 3SL is a potential anti-leukemia agent affecting differentiation and apoptosis.

Rubrofusarin Attenuates Chronic Restraint Stress-Induced Depressive Symptoms.

The aim of this study was to examine whether rubrofusarin, an active ingredient of the Cassia species, has an antidepressive effect in chronic restraint stress (CRS) mouse model. Although acute treatment using rubrofusarin failed, chronic treatment using rubrofusarin ameliorated CRS-induced depressive symptoms. Rubrofusarin treatment significantly reduced the number of Fluoro-Jade B-positive cells and caspase-3 activation within the hippocampus of CRS-treated mice. Moreover, rubrofusarin treatment significantly increased the number of newborn neurons in the hippocampus of CRS-treated mice. CRS induced activation of glycogen synthase kinase-3ß and regulated development and DNA damage responses, and reductions in the extracellular-signal-regulated kinase pathway activity were also reversed by rubrofusarin treatment. Microglial activation and inflammasome markers, including nod-like receptor family pyrin domain containing 3 and adaptor protein apoptosis-associated speck-like protein containing CARD, which were induced by CRS, were ameliorated by rubrofusarin. Synaptic plasticity dysfunction within the hippocampus was also rescued by rubrofusarin treatment. Within in vitro experiments, rubrofusarin blocked corticosterone-induced long-term potentiation impairments. These were blocked by LY294002, which is an Akt inhibitor. Finally, we found that the antidepressant effects of rubrofusarin were blocked by an intracerebroventricular injection of LY294002. These results suggest that rubrofusarin ameliorated CRS-induced depressive symptoms through PI3K/Akt signaling.

REDD1 Is Involved in Amyloid ß-Induced Synaptic Dysfunction and Memory Impairment.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by neurological dysfunction, including memory impairment, attributed to the accumulation of amyloid ß (Aß) in the brain. Although several studies reported possible mechanisms involved in Aß pathology, much remains unknown. Previous findings suggested that a protein regulated in development and DNA damage response 1 (REDD1), a stress-coping regulator, is an Aß-responsive gene involved in Aß cytotoxicity. However, we still do not know how Aß increases the level of REDD1 and whether REDD1 mediates Aß-induced synaptic dysfunction. To elucidate this, we examined the effect of Aß on REDD1-expression using acute hippocampal slices from mice, and the effect of REDD1 short hairpin RNA (shRNA) on Aß-induced synaptic dysfunction. Lastly, we observed the effect of REDD1 shRNA on memory deficit in an AD-like mouse model. Through the experiments, we found that Aß-incubated acute hippocampal slices showed increased REDD1 levels. Moreover, Aß injection into the lateral ventricle increased REDD1 levels in the hippocampus. Anisomycin, but not actinomycin D, blocked Aß-induced increase in REDD1 levels in the acute hippocampal slices, suggesting that Aß may increase REDD1 translation rather than transcription. Aß activated Fyn/ERK/S6 cascade, and inhibitors for Fyn/ERK/S6 or mGluR5 blocked Aß-induced REDD1 upregulation. REDD1 inducer, a transcriptional activator, and Aß blocked synaptic plasticity in the acute hippocampal slices. REDD1 inducer inhibited mTOR/Akt signaling. REDD1 shRNA blocked Aß-induced synaptic deficits. REDD1 shRNA also blocked Aß-induced memory deficits in passive-avoidance and object-recognition tests. Collectively, these results demonstrate that REDD1 participates in Aß pathology and could be a target for AD therapy.

4-O-carboxymethylascochlorin protected against microglial-mediated neurotoxicity in SH-SY5Y and BV2 cocultured cells from LPS-induced neuroinflammation and death by inhibiting MAPK, NF-κB, and Akt pathways.

In our previous studies, structurally similar compounds of ascochlorin and ascofuranone exhibited anti-inflammatory activity. Neural inflammation plays a significant role in the commence and advancement of neurodegenerative diseases. It is not known whether 4-O-carboxymethylascochlorin (AS-6) regulates the initial stage of inflammatory responses at the cellular level in BV2 microglia cells. We here investigated the anti-inflammatory effects of AS-6 treatment in microglia cells with the microglial protection in neurons. We found that the lipopolysaccharide (LPS)-stimulated production of nitric oxide, a main regulator of inflammation, is suppressed by AS-6 in BV2 microglial cells. In addition, AS-6 dose-dependently suppressed the increase in COX-2 protein and messenger RNA levels in LPS-stimulated BV2 cells. Moreover, AS-6 inhibited the expression and secretion of proinflammatory cytokines in BV2 microglial cells. At the intracellular level, AS-6 inhibited LPS-activated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in BV2 microglial cells. AS-6 negatively affected mitogen-activated protein kinases (MAPK) and Akt phosphorylation: Phosphorylated forms of ERK, JNK, p38, and Akt decreased. To check whether AS-6 protects against inflammatory inducer-mediated neurotoxicity, neuronal SH-SY5Y cells were coincubated with BV2 cells in conditioned medium. AS-6 exerted a neuroprotective effect by suppressing microglial activation by LPS or amyloid-ß peptide. AS-6 is a promising suppressor of inflammatory responses in LPS-induced BV2 cells by attenuating NF-κB and MAPKs signaling. AS-6 protected against microglial-mediated neurotoxicity in SH-SY5Y and BV2 cocultured cells from LPS-induced neuroinflammation and death via inhibiting MAPK, NF-κB, and Akt pathways.

Esculentoside H inhibits colon cancer cell migration and growth through suppression of MMP-9 gene expression via NF-kB signaling pathway.

A water-soluble saponin, Esculentoside H (EsH), 3-O-(O-ß-d-glucopyranosyl-(1→4)-ß-d-xylopyranosyl)-28-ß-d-glucopyranosylphytolaccagenin has been isolated and purified from the root extract of perennial plant Phytolacca esculenta. EsH is known to be an anticancer compound, having a capacity for TNF-α release. However, the effects of EsH on migration and growth in tumor cells have not yet been reported. In the current study, the suppressive effects of EsH on phorbol 12-myristate 13-acetate (PMA)-induced cell migration were examined in murine colon cancer CT26 cells and human colon cancer HCT116 cells. Interestingly, the transwell assay and wound healing show that EsH suppresses the PMA-induced migration and growth potential of HCT116 and CT26 colon cancer cells, respectively. EsH dose-dependently suppressed matrix metalloproteinases-9 (MMP-9) expression that was upregulated upon PMA treatment in messenger RNA levels and protein secretion. Since the expression of MMP-9 is correlated with nuclear factor-κB (NF-κB) signaling, it has been examined whether EsH inhibits PMA-induced IκB phosphorylation that leads to the suppression of NK-κB nuclear translocation. EsH repressed the phosphorylation level of JNK, but not extracellular signal-regulated kinase and p38 signaling when the cells were treated with PMA. Overall, these results demonstrated that EsH could suppress cancer migration through blockage of the JNK1/2 and NF-κB signaling-mediated MMP-9 expression.

Ganglioside GM3 suppresses lipopolysaccharide-induced inflammatory responses in rAW 264.7 macrophage cells through NF-κB, AP-1, and MAPKs signaling.

Gangliosides are known to specifically inhibit vascular leukocyte recruitment and consequent interaction with the injured endothelium, the basic inflammatory process. In this study, we have found that the production of nitric oxide (NO), a main regulator of inflammation, is suppressed by GM3 on murine macrophage RAW 264.7 cells, when induced by LPS. In addition, GM3 attenuated the increase in cyclooxyenase-2 (COX-2) protein and mRNA levels in lipopolysaccharide (LPS)-activated RAW 264.7 cells in a dose-dependent manner. Moreover, GM3 inhibited the expression and release of pro-inflammatory cytokines of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß) in RAW 264.7 macrophages. At the intracellular level, GM3 inhibited LPS-induced nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and activator protein (AP)-1 in RAW 264.7 macrophages. We, therefore, investigated whether GM3 affects mitogen-activated protein kinase (MAPK) phosphorylation, a process known as the upstream signaling regulator. GM3 dramatically reduced the expression levels of the phosphorylated forms of ERK, JNK, and p38 in LPS-activated RAW 264.7 cells. These results indicate that GM3 is a promising suppressor of the vascular inflammatory responses and ganglioside GM3 suppresses the LPS-induced inflammatory response in RAW 264.7 macrophages by suppression of NF-κB, AP-1, and MAPKs signaling. Accordingly, GM3 is suggested as a beneficial agent for the treatment of diseases that are associated with inflammation.

Transcriptional Activation of Human GD3 Synthase (hST8Sia I) Gene in Curcumin-Induced Autophagy in A549 Human Lung Carcinoma Cells.

Curcumin, a natural polyphenolic compound isolated from the plant Curcuma longa, is known to induce autophagy in various cancer cells, including lung cancer. In the present study, we also confirmed by LC3 immunofluorescence and immunoblotting analyses that curcumin triggers autophagy in the human lung adenocarcinoma A549 cell line. In parallel with autophagy induction, the gene expression of human GD3 synthase (hST8Sia I) responsible for ganglioside GD3 synthesis was markedly elevated in response to curcumin in the A549 cells. To investigate the transcriptional activation of hST8Sia I associated with the autophagy formation in curcumin-treated A549 cells, functional characterization of the 5'-flanking region of the hST8Sia I gene was carried out using the luciferase reporter assay system. Deletion analysis demonstrated that the -1146 to -646 region, which includes the putative c-Ets-1, CREB, AP-1, and NF-κB binding sites, functions as the curcumin-responsive promoter of hST8Sia I in A549 cells. The site-directed mutagenesis and chromatin immunoprecipitation assay demonstrated that the NF-κB binding site at -731 to -722 was indispensable for the curcumin-induced hST8Sia I gene expression in A549 cells. Moreover, the transcriptional activation of hST8Sia I by the curcumin A549 cells was strongly inhibited by compound C, an inhibitor of AMP-activated protein kinase (AMPK). These results suggest that curcumin controls hST8Sia I gene expression via AMPK signal pathway in A549 cells.

Echinocystic Acid Facilitates Neurite Outgrowth in Neuroblastoma Neuro2a Cells and Enhances Spatial Memory in Aged Mice.

Aging leads to functional changes in the brain and decreases ability of learning and memory. Neurite outgrowth is important in learning and memory, therefore regulation of neurite outgrowth might be a candidate for treating aged brain. Echinocystic acid (EA), a pentacyclic triterpene, has shown to exert various neurological effects. However, the effect of EA on neurite outgrowth has not been studied. In this study, we examined if EA is effective on neurite outgrowth and memory in aged mice. The effect of EA on neurite outgrowth was observed by examining neurite processes of Neuro2a cells treated with EA. Western blot analysis was conducted to examine possible mechanisms. Morris water maze test was used to examine the effect of EA on learning and memory in aged mice. Immunohistochemistry was conducted to observe the effect of EA on neurite outgrowth in the hippocampus. EA was shown to induce neurite outgrowth in a concentration dependent manner without affecting cell viability. Moreover, EA treatment increased phosphorylation of c-jun N-terminal kinase (JNK) and JNK inhibitor, SP600125, blocked the effect of EA on neurite outgrowth. These results demonstrated that EA treatment promotes neurite outgrowth through the JNK signaling pathway. In in vivo experiments, EA treatment increased neurite outgrowth in aged mouse hippocampus. Moreover, EA treatment enhanced spatial learning and memory in aged mice. These results suggest that EA can be developed as a new, naturally occurring drug to treat ageing-related neurological diseases.

The Seed of Zizyphus jujuba var. spinosa Attenuates Alzheimer's Disease-Associated Hippocampal Synaptic Deficits through BDNF/TrkB Signaling.

Ziziphus jujuba is a plant, which bears fruits and seeds that are used for medicinal purposes in Traditional oriental medicine. The seed of Zizyphus jujuba var. spinosa (EZJ) has been also traditionally used for psychiatric disorders in Chinese and Korean medicines. Recent findings have indicated that EZJ improves memory impairment, a common symptom of various neurological diseases. However, the effects of EZJ on amyloid ß (Aß) toxicity, which is a main cause of Alzheimer's disease (AD), remain to be elucidated. To illuminate the potential anti-AD effect and mechanism in the mouse hippocampal tissue, we examined the effect of standardized EZJ on Aß-induced synaptic long-term potentiation (LTP) deficit in the hippocampal tissue. EZJ blocked Aß-induced LTP deficits in a concentration-dependent manner. Moreover, EZJ increased brain-derived neurotrophic factor (BDNF) level in naïve hippocampal slices. The finding that the blockade of BDNF receptor reduced the effect of EZJ suggests that EZJ ameliorates the Aß-induced LTP deficit through BDNF/topomyosin receptor kinase B (TrkB) signaling. However, transcription or translation inhibitors failed to block the effect of EZJ, suggesting that BDNF synthesis is not required for the action of EZJ on LTP. Finally, we found that EZJ stimulates plasmin activity. In contrast, plasmin inhibitor blocked the effect of EZJ on the Aß-induced LTP deficit. Our findings indicate that EZJ ameliorates Aß-induced LTP deficits through BDNF/TrkB signaling. This phenomenon is induced by a regulatory effect of EZJ on the post-translation modification of BDNF.

DISTRIBUTION OF VIRULENCE GENES IN ENVIRONMENTAL AND CLINICAL ISOLATES OF LEGIONELLA PNEUMOPHILA IN BUSAN, SOUTH KOREA.

Legionella pneumophila is the major causative agent of Legionnaires' disease. The distribution of L. pneumophila in environmental water systems of public facilities in Busan, South Korea was previously reported; however, the distribution of virulence genes in environmental and clinical isolates in Busan is unknown. Here, we investigated using PCR the distribution of three virulence loci (dot/icm, lvh, and rtxA) in isolates from Busan. A high prevalence of environmental (127/254) and clinical (3/4) isolates were observed for the simultaneous presence of all three genes, while environmental harboring dot/icm + rtxA, dot/icm + lvh, rtxA + lvh, and only one gene were 21%, 11%, 9% and <1-4%, respectively, The remaining clinical isolate carried dot/icm + rtxA. In addition, 68% of isolates from serogroup 1, the main cause of legionellosis, possessed all three virulence genes compared with 40% of isolates from serogroups 2 to 15. Prevalence of isolates with two genes was 5-14% and 6-29% in serogroup 1 and serogroups 2 to 15, respectively, whereas that of isolates with one gene was 1-2% and 0-6%, respectively. These results provide valuable information for epidemiological investigations of the relationship between environmental and clinical isolates in legionellosis outbreaks in Busan.

Anti-Inflammatory Effect of Ascochlorin in LPS-Stimulated RAW 264.7 Macrophage Cells Is Accompanied With the Down-Regulation of iNOS, COX-2 and Proinflammatory Cytokines Through NF-κB, ERK1/2, and p38 Signaling Pathway.

A natural compound C23 H32 O4 Cl, ascochlorin (ASC) isolated from an incomplete fungus, Ascochyta viciae has been known to have several biological activities as an antibiotic, antifungal, anti-cancer, anti-hypolipidemic, and anti-hypertension agent. In this study, anti-inflammatory activity has been investigated in lipopolysaccharide (LPS)-induced murine macrophage RAW 264.7 cells, since ASC has not been observed on the inflammatory events. The present study has clearly shown that ASC (1-50 µM) significantly suppressed the production of nitric oxide (NO) and prostaglandin E2 (PGE2 ) and decreased the gene expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in a dose-dependent manner. Moreover, ASC inhibited the mRNA expression and the protein secretion of interleukin (IL)-1ß and IL-6 but not tumor necrosis factor (TNF)-α in LPS-stimulated RAW 264.7 macrophage cells. In addition, ASC suppressed nuclear translocation and DNA binding affinity of nuclear factor-κB (NF-κB). Furthermore, ASC down-regulated phospho-extracellular signal-regulated kinase 1/2 (p-ERK1/2) and p-p38. These results demonstrate that ASC exhibits anti-inflammatory effects in RAW 264.7 macrophage cells.

Housekeeping promoter 5'pcmah-2 of pig CMP-N-acetylneuraminic acid hydroxylase gene for NeuGc expression.

In the present study, we isolated pCMAH house-keeping promoter regions (Ph), which are responsible for transcriptional regulation and which are located upstream of the alternative transcript pcmah-2. Luciferase reporter assays using serial construction of each deleted promoter demonstrated that the Ph promoter was highly active in pig-derived kidney PK15. Ph promoter of pcmah lacked a TATA box, but contained three putative Sp1 binding sites. Mutations of these Sp1 binding sites always resulted in the reduction of luciferase activities in Ph-334. In addition, treatment with mithramycin A (25-100 nM) decreased the luciferase activities of the Ph promoters and NeuGc expression in a dose-dependent manner. Electrophoretic mobility shift assay analysis revealed that the probes containing each Sp1 binding site bound to Sp1. Taken together, the results indicate that Sp1 bind to their putative binding sites on the Ph promoter regions of pcmah and positively regulate the promoter activity in pig kidney cells. Interspecies comparison of 5'UTRs and 5'flanking regions shows high homology between pig and cattle, and Sp1 binding sites existing in genomic regions corresponding Ph region are evolutionally conserved.

Upregulation of Human ST8Sia VI (α2,8-Sialyltransferase) Gene Expression by Physcion in SK-N-BE(2)-C Human Neuroblastoma Cells.

In this research, we firstly demonstrated that physcion, an anthraquinone derivative, specifically increased the expression of the human α2,8-sialyltransferase (hST8Sia VI) gene in SK-N-BE(2)-C human neuroblastoma cells. To establish the mechanism responsible for the up-regulation of hST8Sia VI gene expression in physcion-treated SK-N-BE(2)-C cells, the putative promoter region of the hST8Sia VI gene was functionally characterized. Promoter analysis with serially truncated fragments of the 5'-flanking region showed that the region between -320 and -240 is crucial for physcion-induced transcription of hST8Sia VI in SK-N-BE(2)-C cells. Putative binding sites for transcription factors Pax-5 and NF-Y are located at this region. The Pax-5 binding site at -262 to -256 was essential for the expression of the hST8Sia VI gene by physcion in SK-N-BE(2)-C cells. Moreover, the transcription of hST8Sia VI induced by physcion in SK-N-BE(2)-C cells was inhibited by extracellular signal-regulated protein kinase (ERK) inhibitor U0126 and p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580, but not c-Jun N-terminal kinase (JNK) inhibitor SP600125. These results suggest that physcion upregulates hST8Sia VI gene expression via ERK and p38 MAPK pathways in SK-N-BE(2)-C cells.

Exogenous and Endogeneous Disialosyl Ganglioside GD1b Induces Apoptosis of MCF-7 Human Breast Cancer Cells.

Gangliosides have been known to play a role in the regulation of apoptosis in cancer cells. This study has employed disialyl-ganglioside GD1b to apoptosis in human breast cancer MCF-7 cells using exogenous treatment of the cells with GD1b and endogenous expression of GD1b in MCF-7 cells. First, apoptosis in MCF-7 cells was observed after treatment of GD1b. Treatment of MCF-7 cells with GD1b reduced cell growth rates in a dose and time dependent manner during GD1b treatment, as determined by XTT assay. Among the various gangliosides, GD1b specifically induced apoptosis of the MCF-7 cells. Flow cytometry and immunofluorescence assays showed that GD1b specifically induces apoptosis in the MCF-7 cells with Annexin V binding for apoptotic actions in early stage and propidium iodide (PI) staining the nucleus of the MCF-7 cells. Treatment of MCF-7 cells with GD1b activated apoptotic molecules such as processed forms of caspase-8, -7 and PARP (Poly(ADP-ribose) polymerase), without any change in the expression of mitochondria-mediated apoptosis molecules such as Bax and Bcl-2. Second, to investigate the effect of endogenously produced GD1b on the regulation of cell function, UDP-gal: ß1,3-galactosyltransferase-2 (GD1b synthase, Gal-T2) gene has been transfected into the MCF-7 cells. Using the GD1b synthase-transfectants, apoptosis-related signal proteins linked to phenotype changes were examined. Similar to the exogenous GD1b treatment, the cell growth of the GD1b synthase gene-transfectants was significantly suppressed compared with the vector-transfectant cell lines and transfection activated the apoptotic molecules such as processed forms of caspase-8, -7 and PARP, but not the levels of expression of Bax and Bcl-2. GD1b-induced apoptosis was blocked by caspase inhibitor, Z-VAD. Therefore, taken together, it was concluded that GD1b could play an important role in the regulation of breast cancer apoptosis.