Ricky Ngok-Shun Wong

Modulating Angiogenesis The Yin and the Yang in Ginseng

Background—Ginseng is a commonly used nutraceutical. Intriguingly, existing literature reports both wound-healing and antitumor effects of ginseng extract through opposing activities on the vascular system. To elucidate this perplexity, we merged a chemical fingerprinting approach with a deconstructional study of the effects of pure molecules from ginseng extract on angiogenesis. Methods and Results—A mass spectrometric compositional analysis of American, Chinese and Korean, and Sanqi ginseng revealed distinct “sterol ginsenoside” fingerprints, especially in the ratio between a triol, Rg1, and a diol, Rb1, the 2 most prevalent constituents. Using a Matrigel implant model and reconstituting the extracts using distinct ratios of the 2 ginsenosides, we demonstrate that the dominance of Rg1 leads to angiogenesis, whereas Rb1 exerts an opposing effect. Rg1 also promoted functional neovascularization into a polymer scaffold in vivo and the proliferation of, chemoinvasion of, and tubulogenesis by endothelial cells in vitro, an effect mediated through the expression of nitric oxide synthase and the phosphatidylinositol-3 kinase→Akt pathway. In contrast, Rb1 inhibited the earliest step in angiogenesis, the chemoinvasion of endothelial cells. Conclusions—The present study explains, for the first time, the ambiguity about the effects of ginseng in vascular pathophysiology based on the existence of opposing active principles in the extract. We also unraveled a speciogeographic variation impinging on the compositional fingerprint that may modulate the final phenotype. This emphasizes the need for regulations standardizing herbal therapy, currently under the Dietary Supplement and Health Education Act. Furthermore, we propose that Rg1 could be a prototype for a novel group of nonpeptide molecules that can induce therapeutic angiogenesis, such as in wound healing.

Diosgenin induces cell cycle arrest and apoptosis in human leukemia K562 cells with the disruption of Ca2+ homeostasis

PurposeDiosgenin is a steroidal sapogenin with estrogenic and antitumor properties. In order to elucidate the mechanism of its antiproliferative activity, we investigated its effects on the cell cycle and apoptosis in human chronic myelogenous leukemia K562 cells.MethodsCell viability was assessed via an MTT assay. Apoptosis was investigated in terms of nuclear morphology, DNA fragmentation, and phosphatidylserine externalization. Cell cycle analysis was performed via PI staining and flow cytometry (FCM). Western blotting and immunofluorescence methods were used to determine the levels of p53, cell cycle-related proteins and Bcl-2 family members. FCM was also used to estimate the changes in mitochondrial membrane potential (MMP), intracellular Ca2+ concentration and reactive oxygen species (ROS) generation.ResultsCell cycle analysis showed that diosgenin caused G2/M arrest independently of p53. The levels of cyclin B1 and p21Cip1/Waf1 were decreased, whereas cdc2 levels were increased. Subsequent apoptosis was demonstrated with the dramatic activation of caspase-3. A dramatic decline in intracellular Ca2+ concentration was observed as an initiating event in the process of cell cycle arrest and apoptosis, which was followed by the hyperpolarization and depolarization of MMP. Generation of ROS was observed in the progression of apoptosis. The antiapoptotic Bcl-2 and Bcl-xL proteins were downregulated, whereas the proapoptotic Bax was upregulated.ConclusionsDiosgenin inhibits K562 cell proliferation via cell cycle G2/M arrest and apoptosis, with disruption of Ca2+ homeostasis and mitochondrial dysfunction playing vital roles.

Ginsenoside-Rg1 Induces Vascular Endothelial Growth Factor Expression through the Glucocorticoid Receptor-related Phosphatidylinositol 3-Kinase/Akt and β-Catenin/T-cell Factor-dependent Pathway in Human Endothelial Cells

Ginsenoside-Rg1, the most prevalent active constituent of ginseng, is a potent proangiogenic factor of vascular endothelial cells. This suggests that Rg1 may be a new modality for angiotherapy. Rg1 can activate the glucocorticoid receptor (GR). However, the regulatory steps downstream from GR that promote Rg1-induced angiogenesis have not been elucidated. Here we showed for the first time that Rg1 was a potent stimulator of vascular endothelial growth factor (VEGF) expression in human umbilical vein endothelial cells, and importantly this induction was mediated through a phosphatidylinositol 3-kinase (PI3K)/Akt and β-catenin/T-cell factor-dependent pathway via the GR. Rg1 stimulation resulted in an increase in the level of β-catenin, culminating its nuclear accumulation, and subsequent activation of VEGF expression. Transfection of a stable form of β-catenin (S37A) or the use of a glycogen synthase kinase 3β inhibitor to stabilize β-catenin induced VEGF synthesis, whereas small interfering RNA-mediated down-regulation of β-catenin did not, confirming that the effect was β-catenin-specific. Using a luciferase reporter gene assay, we observed that Rg1 increased T-cell factor/lymphoid enhancer factor transcriptional activity. These events were mediated via a PI3K-dependent phosphorylation of the inhibitory Ser9 residue of glycogen synthase kinase 3β. In addition, the GR antagonist RU486 was able to inhibit Rg1-induced PI3K/Akt and β-catenin activation. These findings provide new insights into the mechanism responsible for Rg1 functions.

Signaling pathway of ginsenoside-Rg1 leading to nitric oxide production in endothelial cells

We here provide definitive evidence that ginsenoside‐Rg1, the pharmacologically active component of ginseng, is a functional ligand of the glucocorticoid receptor (GR) as determined by fluorescence polarization assay. Rg1 increased the phosphorylation of GR, phosphatidylinositol‐3 kinase (PI3K), Akt/PKB and endothelial nitric oxide synthase (eNOS) leading to increase nitric oxide (NO) production in human umbilical vein endothelial cell. Rg1‐induced eNOS phosphorylation and NO production were significantly reduced by RU486, LY294,002, or SH‐6. Also, knockdown of GR completely eliminated the Rg1‐induced NO production. This study revealed that Rg1 can indeed serve as an agonist ligand for GR and the activated GR can induce rapid NO production from eNOS via the non‐transcriptional PI3K/Akt pathway.

Role of microRNA-214 in ginsenoside-Rg1-induced angiogenesis.

MicroRNAs (miRNAs) are small non-coding RNAs that act as post-transcriptional gene modulators. Ginsenoside-Rg1, one of the active components of ginseng, has been confirmed as an angiogenesis inducer. Using miRNA microarray analysis, a total of 17 (including miR-214) and 5 miRNAs were found to be down- or up-regulated by Rg1 in human umbilical vein endothelial cells (HUVECs), respectively. Since miR-214 is closely related to endothelial nitric oxide synthase (eNOS) and hence angiogenesis, its expression was further validated by qRT-PCR. We also investigated the role of miR-214 on eNOS expression and in tubulogenesis and motility of HUVEC by transfection of specific miRNA inhibitor or precursor. Our results suggested that Rg1 can down-regulate miR-214 expression in HUVEC, leading to an increase in eNOS expression, and in vitro cell migration and tube formation which can possibly promote angiogenesis. These results signify a new understanding towards how a simple natural compound can affect physiological changes through modulation of miRNA expression.

Neuroprotective effects of ginsenoside-Rg1 in primary nigral neurons against rotenone toxicity

Ginsenoside-Rg1, the pharmacologically active component isolated from ginseng, demonstrated neuroprotective effects on primary cultured rat nigral neurons against rotenone toxicity. Rotenone, a common household pesticide known for its specific and irreversible mitochondria complex I inhibition, has been suggested to be the causal agent of Parkinsons disease (PD) by inducing degeneration of cells in the substantial nigra. The present study demonstrated that co-treatment of rotenone and Rg1 could reduce rotenone-induced cell death by 58% (SEM=+/-5.60; N=3). Rotenone-induced mitochondria membrane potential (MMP, DeltaPsim) depletion was restored and elevated by at least 38% (SEM=+/-2.15; N=3) by Rg1. In addition, Rg1 prevented cytochrome c release from the mitochrondrial membrane and increased the phosphorylation inhibition of the pro-apoptotic protein Bad through activation of the PI3K/Akt pathway. The protective effects of Rg1 was blocked by glucocorticoid receptor antagonist RU486, indicating that the action of Rg1 is mediated through glucocorticoid receptor (GR). In conclusion, Rg1 inhibits the mitochondrial apoptotic pathway and increases the survival chance of the primary cultured nigral neurons against rotenone toxicity. Thus, Rg1 and its related compounds may be developed as protective agents against neurodegenerative diseases induced by mitochondrial toxins.

Ginsenoside Rb1 inhibits tube-like structure formation of endothelial cells by regulating pigment epithelium-derived factor through the oestrogen β receptor

Angiogenesis is a crucial step in tumour growth and metastasis. Ginsenoside‐Rb1 (Rb1), the major active constituent of ginseng, potently inhibits angiogenesis in vivo and in vitro. However, the underlying mechanism remains unknown. We hypothesized that the potent anti‐angiogenic protein, pigment epithelium‐derived factor (PEDF), is involved in regulating the anti‐angiogenic effects of Rb1.

A Simplified Method for Quantifying Cell Migration/Wound Healing in 96-Well Plates

Cell migration plays a key role in both normal physiological and pathological conditions. The study of cell migration and its underlying mechanisms is of great significance in various fields of research, including basic biology and pharmaceutical development. The cell migration or scratch wounding assay is an easy and economical in vitro method that allows researchers to assess a large number of testing compounds. Even though this simple assay has been used for decades, researchers are still trying to modify such experimental protocols and wounding devices. In this study, an 8-channel mechanical “wounder” was designed for performing a cell migration assay, particularly in a 96-well culture plate format. With special designs of a guiding bar and adjustable pins for use with disposable pipette tips, this wounder confined the scratch area within the center of each well to ensure a perfect contact between the pins and the well surface. As a result, this mechanical wounder produces a uniform denudation of a cell monolayer in a 96-well plate with a wound size of around 600 µm. Using this improved wounding device, the effects of epidermal growth factor and DL-α-difluoromethylornithine on the reepithelialization of rat intestinal epithelial cells (IEC-6) and serum on the wound recovery of human umbilical vein endothelial cells were demonstrated. This wounder facilitates cell migration study and can be applicable for multiple sample analysis.

The anti-angiogenic effect of sinomenine

Sinomenine is an alkaloid extracted from the Chinese medicinal plant, Sinomenium acutum, which has been utilized to treat rheumatoid arthritis (RA) in China for over 2000 years. Sinomenine has been shown to mediate a wide range of pharmacological actions which includes anti-inflammatory and anti-rheumatic effects. RA has been classified as a chronic immune-mediated disease that exhibits overlapping manifestation of inflammatory, abnormal cellular and hormonal immune responses with synovial hyperplasia. Since, angiogenesis is recognized to play a critical role in the development of RA and anti-angiogenic therapy has been proposed as a new therapeutic strategy for treatment of RA, we would like to see if sinomenine possesses anti-angiogenic property. In this study, sinomenine inhibited bFGF-induced proliferation of human umbilical vein endothelial cells (HUVEC) and arrested its cell cycle in G1 phase. Sinomenine disrupted tube formation of HUVEC on Matrigel and suppressed the chemotaxis of HUVEC. In addition, sinomenine reduced neovascularization in Matrigel plug assay as well as microvascular outgrowth in rat aorta ring sprouting assay. These results suggest that sinomenine inhibited bFGF-induced angiogenesis in vitro and in vivo. As the leukocytes–endothelial adhesive interactions also play an important role in inflammation, we found that sinomenine reduced the transmigration of granulocytic differentiated HL60 cells across IL-1β activated HUVEC monolayer. Therefore, the inhibition of leukocytes migration across blood vessel walls and the anti-angiogenic effect of sinomenine may contribute towards its therapeutic mechanisms in alleviating the pathogenesis of RA.

A quinolinyl antipyrine based fluorescence sensor for Zn2+ and its application in bioimaging

By incorporating 4-aminoantipyrine moiety onto 8-aminoquinoline with a suitable spacer, a highly selective and sensitive fluorescent Zn2+ sensor, QPA, was designed and constructed. In 25% ACN-HEPES buffer pH 7.0 solution, QPA exhibited 10.6-fold fluorescence enhancement at 500 nm upon addition of Zn2+. The limit of detection (LOD) was calculated to be 1.3 × 10−7 M according to fluorescence titration. The 1 : 1 binding mode of the metal complex was established by combined UV-vis, fluorescence and HRMS spectroscopic method. The membrane permeability of QPA to living cells and bioimaging of Zn2+ are demonstrated.