Ginsenoside-Rb1-Mediated Anti-angiogenesis via Regulating PEDF and miR-33a through the Activation of PPAR-γ Pathway.

Angiogenesis is the formation of new blood vessels from the existing vasculature, which is involved in multiple biological processes, including atherosclerosis, ischemic heart disease, and cancer. Ginsenoside-Rb1 (Rb1), the most abundant ginsenoside isolated form Panax ginseng, has been identified as a promising anti-angiogenic agent via the up-regulation of PEDF. However, the underlying molecular mechanisms still unknown. In the present study, human umbilical vein endothelial cells (HUVECs) were selected to perform in vitro assays. Rb1 (0-20 nM) treatment induced pigment epithelial-derived factor (PEDF) protein expression in concentration and time-dependent manners. Interestingly, it was also demonstrated that the exposure of Rb1 (10 nM) could increase PEDF protein expression without any alteration on mRNA level, suggesting the involvement of posttranscriptional regulation. Furthermore, bioinformatics predictions indicated the regulation of miR-33a on PEDF mRNA 3'-UTR, which was further confirmed by luciferase reporter gene assay and real-time PCR. Over-expression of pre-miR-33a was found to regress partly Rb1-mediated PEDF increment and anti-angiogenic effect in HUVECs. Additionally, Rb1-reduced miR-33a and increased PEDF expression was prevented by pre-incubation with peroxisome proliferator-activated receptor-γ (PPAR-γ) antagonist (GW9662) or transfection with PPAR-γ siRNA in HUVECs. Taken together, our findings demonstrated that Rb1 exerted anti-angiogenic effects through PPAR-γ signaling pathway via modulating miR-33a and PEDF expressions. Thus, Rb1 may have the potential of being developed as an anti-angiogenic agent, however, further appropriate studies are warranted to evaluate the effect in vivo.

Role of G3BP1 in glucocorticoid receptor-mediated microRNA-15b and microRNA-23a biogenesis in endothelial cells.

MicroRNAs (miRNAs) are a family of non-coding RNAs that play crucial roles in regulating various normal cellular responses. Recent studies revealed that the canonical miRNA biogenesis pathway is subject to sophisticated regulation. Hormonal control of miRNA biogenesis by androgen and estrogen has been demonstrated, but the direct effects of the glucocorticoid receptor (GR) on miRNA biogenesis are unknown. This study revealed the role of GR in miRNA maturation. We showed that two GR agonists, dexamethasone and ginsenoside-Rg1 rapidly suppressed the expression of mature miR-15b, miR-23a, and miR-214 in human endothelial cells. RNA pulldown coupled with proteomic analysis identified GTPase-activating protein (SH3 domain) binding protein 1 (G3BP1) as one of the RNA-binding proteins mediating GR-regulated miRNA maturation. Activated GR induced phosphorylation of v-AKT Murine Thymoma Viral Oncogene Homologue (AKT) kinase, which in turn phosphorylated and promoted nuclear translocation of G3BP1. The nuclear G3BP1 bound to the G3BP1 consensus sequence located on primary miR-15b~16-2 and miR-23a~27a~24-2 to inhibit their maturation. The findings from this study have advanced our understanding of the non-genomic effects of GR in the vascular system.

A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate.

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor metastasis. In this assay, cells are grown to form a confluent monolayer and a mechanical wound is created by scratching with a device. Then the migration rate of the cells towards the denuded area can be monitored by imaging. Our 8-channel mechanical wounder is designed to tackle most of the problems associated with the cell migration assay. Firstly, our wounder can be easily sterilized by autoclaving or with common disinfectants. Secondly, the individual adjustable pins allow even contact with the cell culture plate so that sharp and reproducible wounds can be created. Thirdly, the guiding bars on both sides of the wounder ensure consistent wounding position in each well. The use of disposable plastic pipette tips for wounding can further provide better handling of the wounder as well as to minimize cross-contamination. In conclusion, our cell wounder can provide researchers with a user friendly and reproducible device for performing the cell migration assay using the standard 96-well culture plate.

Anti-inflammatory effects of indirubin derivatives on influenza A virus-infected human pulmonary microvascular endothelial cells.

Influenza A virus (IAV) poses global threats to human health. Acute respiratory distress syndrome and multi-organ dysfunction are major complications in patients with severe influenza infection. This may be explained by the recent studies which highlighted the role of the pulmonary endothelium as the center of innate immune cells recruitment and excessive pro-inflammatory cytokines production. In this report, we examined the potential immunomodulatory effects of two indirubin derivatives, indirubin-3'-(2,3-dihydroxypropyl)-oximether (E804) and indirubin-3'-oxime (E231), on IAV (H9N2) infected-human pulmonary microvascular endothelial cells (HPMECs). Infection of H9N2 on HPMECs induced a high level of chemokines and cytokines production including IP-10, RANTES, IL-6, IFN-ß and IFN-γ1. Post-treatment of E804 or E231 could significantly suppress the production of these cytokines. H9N2 infection rapidly triggered the activation of innate immunity through phosphorylation of signaling molecules including mitogen-activated protein kinases (MAPKs) and signal transducer and activator of transcription (STAT) proteins. Using specific inhibitors or small-interfering RNA, we confirmed that indirubin derivatives can suppress H9N2-induced cytokines production through MAPKs and STAT3 signaling pathways. These results underscore the immunomodulatory effects of indirubin derivatives on pulmonary endothelium and its therapeutic potential on IAV-infection.

Ginsenoside-Rg1 induces angiogenesis by the inverse regulation of MET tyrosine kinase receptor expression through miR-23a.

Therapeutic angiogenesis has been implicated in ischemic diseases and wound healing. Ginsenoside-Rg1 (Rg1), one of the most abundant active components of ginseng, has been demonstrated as an angiogenesis-stimulating compound in different models. There is increasing evidence implicating microRNAs (miRNAs), a group of non-coding RNAs, as important regulators of angiogenesis, but the role of microRNAs in Rg1-induced angiogenesis has not been fully explored. In this report, we found that stimulating endothelial cells with Rg1 could reduce miR-23a expression. In silico experiments predicted hepatocyte growth factor receptor (MET), a well-established mediator of angiogenesis, as the target of miR-23a. Transfection of the miR-23a precursor or inhibitor oligonucleotides validated the inverse relationship of miR-23a and MET expression. Luciferase reporter assays further confirmed the interaction between miR-23a and the MET mRNA 3'-UTR. Intriguingly, ginsenoside-Rg1 was found to increase MET protein expression in a time-dependent manner. We further demonstrated that ginsenoside-Rg1-induced angiogenic activities were indeed mediated through the down-regulation of miR-23a and subsequent up-regulation of MET protein expression, as confirmed by gain- and loss-of-function angiogenic experiments. In summary, our results demonstrated that ginsenoside-Rg1 could induce angiogenesis by the inverse regulation of MET tyrosine kinase receptor expression through miR-23a. This study has broadened our understanding of the non-genomic effects of ginsenoside-Rg1, and provided molecular evidence that warrant further development of natural compound as novel angiogenesis-promoting therapy.

Ginsenoside compound K induces apoptosis in nasopharyngeal carcinoma cells via activation of apoptosis-inducing factor.

BACKGROUND: Nasopharyngeal carcinoma (NPC) has a high incidence rate in Southern China. Although there are conventional therapies, the side effects and toxicities are not always tolerable for patients. Recently, the tumoricidal effect of ginsenosides on different cancer cells has been studied. This study aims to investigate the anti-cancer effect of ginsenosides on NPC cells and their underlying mechanism. METHODS: The cytotoxicity of ginsenosides on NPC cell line HK-1 was measured by MTT assay. Apoptosis was detected by propidium iodide staining followed by flow cytometry. A xenograft tumor model was established by injecting nude mice with HK-1 cells. The activation of caspases and apoptosis-inducing factor (AIF) were evaluated by Western blot analysis. Nuclear translocation of AIF was also studied by immunofluorescence staining. Mitochondrial membrane potential was measured by JC-1 dye using flow cytometry. RESULTS: Four ginsenosides, 20 (S)-Rh2, compound K (CK), panaxadiol (PD) and protopanaxadiol (PPD), induced apoptotic cell death in HK-1 cells in a concentration-dependent manner. CK inhibited HK-1 xenograft tumor growth most extensively and depleted mitochondrial membrane potential depolarization and induced translocation of AIF from cytoplasm to nucleus in HK-1 cells. In addition, depletion of AIF by siRNA abolished CK-induced HK-1 cell death. CONCLUSION: Ginsenoside CK-induced apoptosis of HK-1 cells was mediated by the mitochondrial pathway and could significantly inhibit tumor growth in vivo.

Cytoprotective effect of 20S-Rg3 on benzo[a]pyrene-induced DNA damage.

Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon ubiquitously existing in the environment. Its metabolites have been shown to cause DNA damage and cellular dysfunction in humans. Panax ginseng C.A. Meyer is a Chinese medicinal herb, and ginsenosides are the main active constituent of ginseng. Accumulating evidence had indicated that ginseng extract and ginsenosides possess cytoprotective effects. In this study, the protective effect of ginsenosides on BaP-induced DNA damage in human dermal fibroblasts (HDFs) and HepG2 cells was investigated. The genotoxic effect of BaP was measured by the comet assay. Results showed that tail moment was increased in BaP-treated cells, but cotreatment of ginsenoside 20(S)-Rg3 can significantly decrease BaP-induced DNA damage. A downstream mechanistic study revealed that 20(S)-Rg3 increased the gene expression of an important phase II detoxifying enzyme NAD(P)H:quinine oxidoreductase 1. The effect was also associated with the activation of protein kinase B (Akt) and nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2). These results indicated that 20(S)-Rg3 might protect HDFs from BaP-induced DNA damage through the activation of the phosphatidylinositol 3-kinase/Akt/Nrf2 pathway. Our results also demonstrated that 20(S)-Rg3 is a functional ligand of pregnane X receptor (PXR), a nuclear receptor that mediates the induction of drug clearance pathways. Subsequent knockdown of PXR expression by small interfering RNA confirmed the involvement of PXR on the protective effects of 20(S)-Rg3 against BaP-induced DNA damage. In summary, ginsenoside 20(S)-Rg3 can protect against BaP-induced genotoxicity in human cells, suggesting that ginseng may serve as a natural cytoprotective agent against environmental carcinogens.