Nai-Ki Mak

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.

Cellular uptake, subcellular localization and photodamaging effect of Temoporfin (mTHPC) in nasopharyngeal carcinoma cells: comparison with hematoporphyrin derivative

Temoporfin (meta-tetra (hydroxyphenyl)chlorin; mTHPC) potentiated a 100-fold higher cytotoxic effect than hematoporphyrin derivative (HPD) on two nasopharyngeal carcinoma cell lines (HK1 and CNE2) in terms of the overall photodynamic therapy (PDT) dose. The cellular uptake, evaluated by flow cytometry and spectrophotometry demonstrated that mTHPC exhibited higher uptake ability than HPD. Confocal laser scanning microscopy detection for both the sensitizer and mitochondria probe on the same cell images revealed that both drugs accumulated diffusely in the cytoplasm and that mitochrondria is a target organelle. Photo-activation ruptured the mitochrondria, with more pronounced mitochondrial damage being observed in mTHPC-PDT course. This correlated well with the cell photokilling efficiency of mTHPC.

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.

Degradation of diphenylamine by persulfate: Performance optimization, kinetics and mechanism

The degradation of diphenylamine (DPA) in aqueous solution by persulfate is investigated. Effects of pH, persulfate concentration, ionic strength, temperature and catalytic ions Fe(3+) and Ag(+) on the degradation efficiency of DPA by persulfate are examined in batch experiments. The degradation of DPA by persulfate is found to follow the pseudo-first-order kinetic model. Increasing the reaction temperature or persulfate concentration may significantly accelerate the DPA degradation. Fe(3+) and Ag(+) ions can enhance the degradation of DPA, and Ag(+) ion is more efficient than Fe(3+) ion. However, the increase of either the pH value or ionic strength will decrease the rate of DPA degradation. N-Phenyl-4-quinoneimine, N-carboxyl-4-quinoneimine, 4-quinoneimine and oxalic acid are identified as the major intermediates of DPA degradation, and a primary pathway for the degradation of DPA is proposed. The degradation of DPA in surface water, groundwater and seawater is also tested by persulfate, and more than 90% of DPA can be degraded at room temperature in 45min at an initial concentration of 20mgL(-1).

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.

Comparative Studies of the Cellular Uptake, Subcellular Localization, and Cytotoxic and Phototoxic Antitumor Properties of Ruthenium(II)-Porphyrin Conjugates with Different Linkers

Six water-soluble free-base porphyrin-Ru(II) conjugates, 1-3, and Zn(II) porphyrin-Ru(II) conjugates, 4-6, with different linkers between the hydrophobic porphyrin moiety and the hydrophilic Ru(II)-polypyridyl complex, have been synthesized. The linear and two-photon-induced photophysical properties of these conjugates were measured and evaluated for their potential application as dual in vitro imaging and photodynamic therapeutic (PDT) agents. Conjugates 1-3, with their high luminescence and singlet oxygen quantum yields, were selected for further study of their cellular uptake, subcellular localization, and cytotoxic and photocytotoxic (under linear and two-photon excitation) properties using HeLa cells. Conjugate 2, with its hydrophobic phenylethynyl linker, was shown to be highly promising for further development as a bifunctional probe for two-photon (NIR) induced PDT and in vitro imaging. Cellular uptake and subcellular localization properties were shown to be crucial to its PDT efficacy.

An amphiphilic ruthenium(II)-polypyridyl appended porphyrin as potential bifunctional two-photon tumor-imaging and photodynamic therapeutic agent.

An amphiphilic porphyrin appended with a Ru(II)-polypyridyl complex (Ru-P) showing a moderate two-photon absorption cross-section (178.0+/-26.8GM), high singlet oxygen quantum yield and rapid cellular uptake was synthesized. In vitro study using human nasopharyngeal carcinoma cells showed that Ru-P exhibited a strong two-photon induced fluorescence upon uptake, lysosomal localization and potent two-photon induced cytotoxicity. These results show that Ru-P, which was designed to enhance its cellular uptake, can potentially be used as an efficacious bifunctional two-photon tumor-imaging and photodynamic therapeutic agent despite its moderate two-photon absorption cross-section.