Kyungsun Choi

Ginsenosides compound K and Rh2 inhibit tumor necrosis factor-α-induced activation of the NF-κB and JNK pathways in human astroglial cells

Ginsenosides, the main component of Panax ginseng, have been known for the anti-inflammatory and anti-proliferative activities. In this study, we investigated the molecular mechanisms responsible for the anti-inflammatory effects of ginsenosides on activated astroglial cells. Among 13 different ginsenosides, intestinal bacterial metabolites Rh(2) and compound K (C-K) showed a significant inhibitory effect on tumor necrosis factor-alpha (TNF-alpha)-induced expression of intercellular adhesion molecule-1 in human astroglial cells. Pretreatment with C-K or Rh(2) suppressed TNF-alpha-induced phosphorylation of IkappaBalpha kinase and the subsequent phosphorylation and degradation of IkappaBalpha. Additionally, the same treatment inhibited TNF-alpha-induced phosphorylation of MKK4 and the subsequent activation of the JNK-AP-1 pathway. The inhibitory effect of ginsenosides on TNF-alpha-induced activation of the NF-kappaB and JNK pathways was not observed in human monocytic U937 cells. These results collectively indicate that ginsenoside metabolites C-K and Rh(2) exert anti-inflammatory effects by the inhibition of both NF-kappaB and JNK pathways in a cell-specific manner.

Reactive oxygen species mediate chloroquine‐induced expression of chemokines by human astroglial cells

We have previously demonstrated that chloroquine may evoke inflammatory responses in the central nervous system by inducing expression of pro‐inflammatory cytokines by astroglial cells. In this study, we further examined the molecular mechanism responsible for chloroquine‐induced activation of NF‐κB and subsequent expression of chemokines by astroglial cells. We observed that (1) chloroquine induced expression of chemokines such as CCL2 and CXCL8 in a dose‐ and time‐dependent manner in human astroglial cells; (2) other lysosomotropic agents such as ammonium chloride and bafilomycin A1 had minimal effects on chemokine expression; (3) inhibition of NF‐κB by MG‐132 and TPCK suppressed chloroquine‐induced mRNA expression of chemokines; (4) chloroquine increased the intracellular level of reactive oxygen species (ROS) in a dose‐ and time‐dependent manner by human astroglial cells, but not by monocytic/microglial cells; (5) chloroquine‐induced increase of intracellular ROS level was suppressed by pre‐incubation with diphenyl iodonium (DPI) and N‐acetyl cysteine (NAC); and (6) inhibition of chloroquine‐induced ROS production by DPI or NAC suppressed chloroquine‐mediated activation of NF‐κB and subsequent mRNA expression of chemokines in astroglial cells. These results collectively suggest that chloroquine generates ROS, which is responsible for NF‐κB activation and subsequent expression of pro‐inflammatory chemokines in human astroglial cells.

Hepatitis C virus infection enhances TNFα-induced cell death via suppression of NF-κB†

Hepatitis C virus (HCV) infection results in liver injury and long‐term complications, such as liver cirrhosis and hepatocellular carcinoma. Liver injury in HCV infection is believed to be caused by host immune responses, not by viral cytopathic effects. Tumor necrosis factor‐alpha (TNF‐α) plays a pivotal role in the inflammatory processes of hepatitis C. TNF‐α induces cell death that can be ameliorated by nuclear factor kappaB (NF‐κB) activation. We investigated the regulation of TNF‐α signal transduction in HCV‐infected cells and identified HCV proteins responsible for sensitization to TNF‐α‐induced cell death. We studied the effect of HCV infection on TNF‐α signal transduction using an in vitro HCV infection model (JFH‐1, genotype 2a) with Huh‐7 and Huh‐7.5 cells. We found that TNF‐α‐induced cell death significantly increased in HCV‐infected cells. HCV infection diminished TNF‐α‐induced phosphorylation of IκB kinase (IKK) and inhibitor of NF‐κB (IκB), which are upstream regulators of NF‐κB activation. HCV infection also inhibited nuclear translocation of NF‐κB and expression of NF‐κB‐dependent anti‐apoptotic proteins, such as B‐cell lymphoma—extra large (Bcl‐xL), X‐linked inhibitor of apoptosis protein (XIAP), and the long form of cellular‐FLICE inhibitory protein (c‐FLIP). Decreased levels of Bcl‐xL, XIAP, and c‐FLIP messenger RNA and protein were also observed in livers with chronic hepatitis C. Transfection with plasmids encoding each HCV protein revealed that core, nonstructural protein (NS)4B, and NS5B attenuated TNF‐α‐induced NF‐κB activation and enhanced TNF‐α‐induced cell death. Conclusion: HCV infection enhances TNF‐α‐induced cell death by suppressing NF‐κB activation through the action of core, NS4B, and NS5B. This mechanism may contribute to immune‐mediated liver injury in HCV infection. (HEPATOLOGY 2012;56:831–840)

Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein–protein interaction module

Nanoparticle-mediated delivery of functional macromolecules is a promising method for treating a variety of human diseases. Among nanoparticles, cell-derived exosomes have recently been highlighted as a new therapeutic strategy for the in vivo delivery of nucleotides and chemical drugs. Here we describe a new tool for intracellular delivery of target proteins, named ‘exosomes for protein loading via optically reversible protein–protein interactions’ (EXPLORs). By integrating a reversible protein–protein interaction module controlled by blue light with the endogenous process of exosome biogenesis, we are able to successfully load cargo proteins into newly generated exosomes. Treatment with protein-loaded EXPLORs is shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in vitro and in vivo. These results clearly indicate the potential of EXPLORs as a mechanism for the efficient intracellular transfer of protein-based therapeutics into recipient cells and tissues. Exosomes have been identified as promising vehicles for the in vivodelivery of therapeutic molecules. Here the authors design a system to load protein cargos into exosomes during their biogenesis using optogenetic control of protein-protein interactions between the cargo and an exosome-localized partner.

Multiplex analysis of cytokines in the serum and cerebrospinal fluid of patients with Alzheimer's disease by color-coded bead technology.

Background and purpose The availability and promise of effective treatments for neurodegenerative disorders are increasing the importance of early diagnosis. Having molecular and biochemical markers of Alzheimers disease (AD) would complement clinical approaches, and further the goals of early and accurate diagnosis. Combining multiple biomarkers in evaluations significantly increases the sensitivity and specificity of the biochemical tests. Methods In this study, we used color-coded bead-based Luminex technology to test the potential of using chemokines and cytokines as biochemical markers of AD. We measured the levels of 22 chemokines and cytokines in the serum and cerebrospinal fluid (CSF) of 32 de novo patients (13 controls, 11 AD, and 8 Parkinsons disease [PD]). Results MCP-1 was the only cytokine detectable in CSF, and its levels did not differ between control and disease groups. However, the serum concentration of eotaxin was significantly higher in AD patients than in the control group. Conclusions The analysis of multiple inflammatory mediators revealed marginal differences in their CSF and serum concentrations for the differential diagnosis of AD and PD. These results provide evidence that immunological responses are not major contributors to the pathogenesis of AD and PD.

Hydrogen peroxide enhances TRAIL-induced cell death through up-regulation of DR5 in human astrocytic cells.

The central nervous system (CNS) is particularly vulnerable to reactive oxygen species (ROS), which have been implicated in the pathogenesis of various neurological disorders. The TNF superfamily of cytokines, especially tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), induces caspase-dependent cell death and is also implicated in various neurodegenerative diseases. In this study, we investigated the relationship between ROS and TRAIL-induced cell death. Exposure to hydrogen peroxide (H(2)O(2)) (100 microM) sensitized human astrocytic cells to TRAIL-induced cell death (up to 7-fold induction). To delineate the molecular mechanisms responsible for H(2)O(2)-induced sensitization, we examined expression of various genes (Caspase-8, Fas, FasL, DR4, DR5, DcR1, DcR2, TRAIL, TNFRp55) related to TRAIL-induced cell death. Treatment with H(2)O(2) significantly increased DR5 mRNA and protein expression in a time- and dose-dependent manner. H(2)O(2)-mediated cell death was blocked upon treatment with DR5:Fc protein, a TRAIL-specific antagonistic protein. These findings collectively suggest that oxidative stress sensitizes human astroglial cells to TRAIL-induced cell death through up-regulation of DR5 expression.

Proapoptotic Ginsenosides Compound K and Rh2 Enhance Fas-induced Cell Death of Human Astrocytoma Cells Through Distinct Apoptotic Signaling Pathways

PURPOSE Malignant astrocytomas are among the commonest primary brain tumors and they have a grave prognosis, and so there is an urgent need to develop effective treatment. In this study, we investigated the molecular mechanisms that are responsible for the anti-tumor effect of ginsenosides on human astrocytoma cells. MATERIALS AND METHODS We tested 13 different ginsenosides for their anti-tumor effect on human malignant astrocytoma cells in conjunction with Fas stimulation. In addition, the cell signaling pathways were explored by using pharmacological inhibitors and performing immunoblot analysis. DCF-DA staining and antioxidant experiments were performed to investigate the role of reactive oxygen species as one of the apoptosis-inducing mechanisms. RESULTS Among the 13 different ginsenoside metabolites, compound K and Rh(2) induced apoptotic cell death of the astrocytoma cells in a caspase- and p38 MAPK-dependent manner, yet the same treatment had no cytotoxic effect on the primary cultured human astrocytes. Combined treatment with ginsenosides and Fas ligand showed a synergistic cytotoxic effect, which was mediated by the reduction of intracellular reactive oxygen species. CONCLUSION These results suggest that ginsenoside metabolites in combination with Fas ligand may provide a new strategy to treat malignant astrocytomas, which are tumors that are quite resistant to conventional anti-cancer treatment.

Phenotypic Modulation of Primary Vascular Smooth Muscle Cells by Short-Term Culture on Micropatterned Substrate

Loss of contractility and acquisition of an epithelial phenotype of vascular smooth muscle cells (VSMCs) are key events in proliferative vascular pathologies such as atherosclerosis and post-angioplastic restenosis. There is no proper cell culture system allowing differentiation of VSMCs so that it is difficult to delineate the molecular mechanism responsible for proliferative vasculopathy. We investigated whether a micropatterned substrate could restore the contractile phenotype of VSMCs in vitro. To induce and maintain the differentiated VSMC phenotype in vitro, we introduced a micropatterned groove substrate to modulate the morphology and function of VSMCs. Later than 7th passage of VSMCs showed typical synthetic phenotype characterized by epithelial morphology, increased proliferation rates and corresponding gene expression profiles; while short-term culture of these cells on a micropatterned groove induced a change to an intermediate phenotype characterized by low proliferation rates, increased migration, a spindle-like morphology associated with cytoskeletal rearrangement and expression of muscle-specific genes. Microarray analysis showed preferential expression of contractile and smooth muscle cell-specific genes in cells cultured on the micropatterned groove. Culture on a patterned groove may provide a valuable model for the study the role of VSMCs in normal vascular physiology and a variety of proliferative vascular diseases.

Differential dependency of human cancer cells on vascular endothelial growth factor-mediated autocrine growth and survival.

Analysis using the public microarray database Gene Expression Omnibus indicates significantly higher mRNA expression of VEGF and VEGFRs in colorectal cancer and high grade astrocytoma but not in hepatocellular carcinoma compared to normal tissue. Human malignant astrocytoma cell lines (U251-MG and U373-MG) and HT-1080 fibrosarcoma cells expressed relatively higher levels of VEGF and VEGFRs compared to hepatocellular and colorectal cancer cell lines. Administration of exogenous VEGF-A induced cell growth in a dose-dependent fashion in astrocytoma and fibrosarcoma cells but not in colorectal and hepatocellular cancer cells. The blockade of VEGF inhibited cell survival only in U251-MG, U373-MG and HT-1080 cells. These results collectively suggest the role of autocrine VEGF signaling in various cancer cells and provide a basis for the variable clinical responses to antiangiogenic therapy observed in different types of malignancies.

Blockade of VEGF-A suppresses tumor growth via inhibition of autocrine signaling through FAK and AKT

Blockade of VEGF signaling using RNA interferences, a neutralizing antibody, an antagonizing soluble VEGF receptor, and a receptor tyrosine kinase inhibitor induced anti-tumor effects in human astrocytoma U251-MG and fibrosarcoma HT-1080 in vitro in a dose-dependent manner. Furthermore, blockade of VEGF-A using the doxycycline-inducible VEGF-A RNA interference system showed a significant anti-tumor effect in a murine HT-1080-xenograft model. Anti-tumor effect through the blockade of VEGF signaling was mediated by FAK and AKT pathway in vitro and in vivo. These results collectively indicate that VEGF-A and its receptors can act as key inducer of tumor growth as well as angiogenesis.