Yeo-Jin Choi

Ginsenoside Rh1 suppresses matrix metalloproteinase-1 expression through inhibition of activator protein-1 and mitogen-activated protein kinase signaling pathway in human hepatocellular carcinoma cells

Invasion and metastasis are the major causes of treatment failure in patients with cancer. Here, we investigated the effects of ginsenoside Rh1 on tumor invasion and metastasis in human hepatocellular carcinoma HepG2 cells and its possible mechanism of action. Rh1 showed concentration- and time-dependent inhibition of HepG2 cell migration and invasion. Matrix metalloproteinase-1 (MMP-1) gene expression and its promoter activity were also concentration-dependently inhibited by Rh1 treatment. The inhibitory effect of Rh1 on MMP-1 expression was due to inactivation of the mitogen-activated protein kinases (MAPKs) ERK, JNK, and p38 MAPK. By transient transfection analysis with the MMP-1 promoter (-2846 to -29 nt) and AP-1 promoter, MMP-1 and AP-1 promoter activities were induced by phorbol myristate acetate (PMA) but were significantly inhibited by PD98059 (ERK1/2 inhibitor) or SP600125 (JNK inhibitor). The induction of MMP-1 and AP-1 promoters by PMA was attenuated by Rh1, and both promoter activities were synergistically inhibited by co-treatment with PD98059. To evaluate the effects of Rh1 on AP-1 dimers, expression analysis and electrophoretic mobility shift (EMSA) assay using radiolabeled AP-1-specific oligomers at proximal site (-73 nt) and distal site (-1600 nt) of the MMP-1 promoter were performed. The results showed that Rh1 inhibited the expression of c-Jun and c-Fos but did not affect the DNA binding ability of AP-1-specific oligomers. However, Rh1 attenuated the stability of c-Jun. Therefore, Rh1 has potential for development of novel chemotherapeutic agents for treatment of malignant cancers, including early hepatocellular carcinoma related to MMP-1 expression.

Anti-metastatic effects of ginsenoside Rd via inactivation of MAPK signaling and induction of focal adhesion formation.

Ginsenoside Rd is a protopanaxadiol-type ginsenoside found in ginseng and is the active ingredient in several Oriental herbal medicines. We investigated the effects of ginsenoside Rd on tumor invasion and metastasis in the human hepatocellular carcinoma HepG2 and its possible mechanism of action. HepG2 cells were treated with ginsenoside Rd at different concentrations. Scratch wound and Boyden chamber assays were used to determine the effects of ginsenoside Rd on the migration and invasiveness of HepG2 cells, respectively. The molecular mechanisms by which ginsenoside Rd inhibited the invasion and migration of HepG2 cells were investigated by RT-PCR, Western blotting, gelatin zymography, promoter assay, and treatment with inhibitors of MAPK signaling. Immunofluorescence analysis was conducted to evaluate the effect of ginsenoside Rd on focal adhesion formation in HepG2 cells. Treatment with ginsenoside Rd dose- and time-dependently inhibited the migration and invasion of HepG2 cells. It achieved this by reducing the expression of MMP-1, MMP-2, and MMP-7, by blocking MAPK signaling by inhibiting the phosphorylation of ERK and p38 MAPK, by inhibition of AP-1 activation, and by inducing focal adhesion formation and modulating vinculin localization and expression. Treatment of HepG2 cells with ginsenoside Rd significantly inhibited metastasis, most likely by blocking MMP activation and MAPK signaling pathways involved in cancer cell migration. These findings may be useful for the development of novel chemotherapeutic agents for the treatment of malignant cancers.

The DEAD-box RNA helicase DDX3 interacts with DDX5, co-localizes with it in the cytoplasm during the G2/M phase of the cycle, and affects its shuttling during mRNP export.

DDX3 is involved in RNA transport, translational control, proliferation of RNA viruses, and cancer progression. From yeast two‐hybrid screening using the C‐terminal region of DDX3 as a bait, the DEAD‐box RNA helicase DDX5 was cloned. In immunofluorescence analysis, DDX3 and DDX5 were mainly co‐localized in the cytoplasm. Interestingly, cytoplasmic levels of DDX5 increased in the G2/M phase and consequently protein–protein interaction also increased in the cytoplasmic fraction. DDX3 was highly phosphorylated at its serine, threonine, and tyrosine residues in the steady state, but not phosphorylated at the serine residue(s) in the G2/M phase. DDX5 was less phosphorylated in the G1/S phase; however, it was highly phosphorylated at serine, threonine, and tyrosine residues in the G2/M phase. PP2A treatment of the cytoplasmic lysate from G2/M phase cells positively affected the interaction between DDX3 and DDX5, whereas, PTP1B treatment did not. In an analysis involving recombinant His‐DDX3 and His‐DDX5, PP2A pretreatment of His‐DDX5 increased the interaction with endogenous DDX3, and vice versa. Furthermore, the results of GST pull‐down experiments support the conclusion that dephosphorylation of serine and/or threonine residues in both proteins enhanced protein–protein interactions. UV cross‐linking experiments showed that DDX3 and DDX5 are involved in mRNP export. Additionally, DDX3 knockdown blocked the shuttling of DDX5 to the nucleus. These data demonstrate a novel interaction between DDX3 and DDX5 through the phosphorylation of both proteins, especially in the G2/M phase, and suggest a novel combined mechanism of action, involving RNP remodeling and splicing, for DEAD‐box RNA helicases involved in mRNP export. J. Cell. Biochem. 113: 985–996, 2012.

Stimulation of TRAF6/TAK1 degradation and inhibition of JNK/AP-1 signalling by ginsenoside Rg3 attenuates hepatitis B virus replication.

In present study, we investigated the effect of ginsenoside Rg3 on hepatitis B virus DNA replication and secretion of hepatitis B surface antigen and e antigen in HepG2.2.15 cells. Rg3 dose- and time-dependently inhibited hepatitis B surface antigen, e antigen, and hepatitis B viral particle secretion. To explore the effect of Rg3 on anti-hepatitis B activity, we analysed toll-like receptor-myeloid differentiation primary response gene 88 signalling. Rg3 did not affect the expression of toll-like receptors or myeloid differentiation primary response gene 88. However, it significantly inhibited the expression of TNF receptor-associated factor 6 and transforming growth factor β activated kinase-1, which are adaptor molecules that signal through a toll-like receptor-myeloid differentiation primary response gene 88-dependent pathway. The inhibitory effect of Rg3 on TNF receptor-associated factor 6/transforming growth factor β activated kinase-1 expression was caused by the downregulation of TNF receptor-associated factor 6 expression as well as the stimulation of ubiquitination and proteasomal degradation of TNF receptor-associated factor 6, followed by downregulation of transforming growth factor β activated kinase-1. Furthermore, Rg3 inhibited mitogen-activated protein kinase signalling by inhibiting c-Jun N-terminal kinase phosphorylation, reduced the expression of AP-1 transcription factors (especially c-Jun and JunB), and inhibited AP-1 promoter activity. The inhibitory effect of Rg3 on c-Jun N-terminal kinase/AP-1 signalling showed anti-inflammatory activity based on the reduction in the expression of pro-inflammatory cytokines, IL-8 and TNF-α, at both the transcriptional and translational levels. Therefore, Rg3 showed anti-hepatitis B activity via the degradation of TNF receptor-associated factor 6/transforming growth factor β activated kinase-1 and the inhibition of c-Jun N-terminal kinase/AP-1 signalling.

Cytoprotective activities of hydroxycinnamic acid amides of serotonin against oxidative stress-induced damage in HepG2 and HaCaT cells

Hydroxycinnamic acid amides of serotonin (HCAAS) [caffeoylserotonin (compound 1), p-coumaroylserotonin (compound 2), and feruloylserotonin (compound 3)] are secondary metabolites produced in plants. The aim of this study was to investigate the cytoprotective effects of HCAAS based on intracellular reactive oxygen radical (ROS) generation, lipid peroxidation, protein carbonylation, and phosphorylation of histone H2AX in H(2)O(2) treated-HepG2 and HaCaT cells. We have shown that HCAAS showed various strong antioxidant activities in hydrogen peroxide treated both cell lines, suggesting that these compounds may play as chemotherapeutic agents for preventing or reducing the oxidative stress-induced diseases.

Ginsenoside Rh1 inhibits the invasion and migration of THP-1 acute monocytic leukemia cells via inactivation of the MAPK signaling pathway.

Ginsenoside Rh1 has been reported to possess antiallergic and anti-inflammatory activities, but its effects on monocytes remain to be determined. Herein, we investigated the effects of Rh1 on the expression of MCP-1 and CCR2, activation of MAPK signaling, and chemotaxis of monocytes. Treatment of Rh1 decreased the levels of MCP-1 and CCR2 and the expression of VLA5 and activated β1 integrin on the cell surface, and attenuated the phosphorylation of MAPKs. Based on these results, the inhibitory effects of Rh1 on monocyte function should be regarded as a promising new anti-inflammatory response with a potential therapeutic role against inflammation-dependent diseases.

Stimulation of DDX3 Expression by Ginsenoside Rg3 through the Akt/p53 Pathway Activates the Innate Immune Response via TBK1/IKKε/IRF3 Signalling

DEAD-box RNA helicase DDX3 is a well-known host factor that inhibits hepatitis B viral proliferation and boosts innate immune responses via TANK-binding kinase 1 (TBK1)/IKKε-mediated and/or interferon (IFN)-β promoter stimulator-1 (IPS-1)-mediated IFN-β induction. Previously, we demonstrated the anti-hepatitis B activity of Rg3 via stimulation of TRAF6/TAK1 degradation and inhibition of JNK/AP-1 signaling. To determine the effects of Rg3 on innate immunity, an IFN-β promoter assay was performed. Rg3 ameliorated IFN-β expression via upregulation of both the TBK1/IKKε pathway and DDX3 expression. In addition, Rg3 induced the phosphorylation of IRF3 and its translocation into nucleus, which is a key molecule to induction of IFN-β expression. To evaluate the molecular mechanism of Rg3 on DDX3 expression, the DDX3 promoter (-1406/+105) was subjected to luciferase assay and ChIP analysis. p53 phosphorylation resulted in upregulation of DDX3 expression, which enhanced DDX3 promoter transactivation activity. Transient transfection with wild-type p53 increased DDX3 promoter activity in Hep3B cells which have null mutant of p53, whereas knockdown p53 by si-p53 reduced DDX3 promoter activity in HepG2.2.15 and HepG2 cells, respectively. Rg3- mediated phosphorylation of p53 resulted in inhibition of Akt phosphorylation, which in turn reduced MDM2-mediated p53 degradation. An Akt inhibitor augmented DDX3 promoter activity and reduced the secretion of hepatitis B surface antigen. Our data indicate that Rg3 enhances innate immunity by inducing IFN-β expression through upregulation of DDX3 promoter activity via p53-mediated transactivation and activation of the TBK1/IKKε/IRF3 pathway.

Comparative Analysis of the Antioxidant Activities of Green Peppers Cultivated under Conventional and Environmental-Friendly Farming conditions

Environmental-friendly agriculture (EFA) is defined as the cultivation of crops with reduced amounts or without chemical-synthetic pesticides. Recently, the use of chemical pesticides has decreased significantly; therefore, we cultivated peppers following EFA- and conventional methods and compared their antioxidant activities. To accomplish this, the environmental-friendly cultivated peppers (EFPE) and conventionally cultivated peppers (CCPE) were extracted with 70% methanol and the effects of the extracts on the cell viability, intracellular ROS generation, lipid peroxidation and catalase activity of HepG2 cells were evaluated. EFPE showed a stronger protective effect against oxidative stress induced-cell death than that of CCPE. EFPE also reduced intracellular ROS generation (42.7% to 26.4%) following treatment with hydrogen peroxide more effectively than that of CCPE (24.2% to 6.3%). Furthermore, EFPE and CCPE showed protective effects against lipid peroxidation and induced catalase activity, although these effects were not statistically significant. Taken together, these results suggest that EFPE showed stronger antioxidant activities than CCPE, and thus represent evidence that EFA with biocontrol materials may improve the functional properties of crops and/or secondary metabolites with antioxidant activities when compared with conventional agricultural practices.