Search

Oseltamivir phosphate (OP, Tamiflu®) is a widely used prodrug for the treatment of influenza viral infections. Orally administered OP is rapidly hydrolyzed by the carboxylesterases in animals to oseltamivir carboxylate (OC), a potent influenza virus neuraminidase inhibitor. The goals of this study were to develop and validate a physiologically-based pharmacokinetic (PBPK) model of OP/OC in rats and humans, and to predict the internal tissue doses for OP and OC in humans after receiving OP orally. To this end, a PBPK model of OP/OC was first developed in the rat, which was then scaled up to humans by replacing the physiological and biochemical parameters with human-specific values. The proposed PBPK model consisted of an OP and an OC sub-models each containing nine first-order, flow-limited tissue/organ compartments. OP metabolism to OC was assumed to carry out mainly by hepatic carboxylesterases although extra-hepatic metabolism also occurred especially in the plasma. The PBPK model was developed and validated by experimental data from our laboratories and from the literature. The proposed PBPK model accurately predicted the pharmacokinetic behavior of OP and OC in humans and rats after receiving a single or multiple doses of OP orally or an OC dose i.v. The PBPK model was used to predict the internal tissue doses of OP and OC in a hypothetical human after receiving the recommended dose of 75 mg/kg OP b.i.d. for 6 days. Steady-state OC concentrations in the plasma and major organs such as the lung and the brain were higher than the minimum in vitro IC50 reported for H1N1 influenza virus neuraminidase, confirming OP is an effective, anti-viral agent. OP side-effects in the gastrointestinal tract and brain of humans were explainable by the tissue doses found in these organs. The PBPK model provides a quantitative tool to evaluate the relationship between an externally applied dose of OP and the internal tissue doses in humans. As such the model can be used to adjust the dose regimens for adult patients in disease states e.g., renal failure and liver damage.

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.

Background: The development of antiangiogenic agents arises as a more effective and selective therapeutic approach for the treatment of cancer. In addition to reduced acute toxicity, the efficacy of chemotherapy could be improved when administered in combination specific antiangiogenic with cytotoxic agents. The conjugation or hybridization of bifunctional molecules is one of the alternative rational design strategies for co-administration of anticancer drugs. Objective and Methods: The goal of this work is to prepare the conjugates of an antiangiogenic triterpene, 3-oxo oleanolic acid, and structurally related triterpenoids with a cytotoxic semibenzoquinone, jacaranone. The cytotoxic, antiproliferative and antiangiogenic activities of segments and conjugates were determined. The possible targets of conjugates 6a-6h were predicted using Similarity Ensemble Approach (SEA). Results: The results showed that these conjugates are more potent in both cytotoxic and antiangiogenic assays than their corresponding parent molecules, and are also selectively more active against melanoma cells B16 and metastatic B16BL6 than the two other cancer cell lines (A549 and MCF-7) tested. The predicted antiangiogenesis related targets could involve glycogen phosphorylase, neuraminidase, interferon gamma, and tubulin beta chain. Conclusion: The bifunctional conjugates could be useful as dual acting antitumor/antigiogenic agents.

Background Our previous studies have demonstrated that ginsenoside-Rg1 can promote angiogenesis in vitro and in vivo through activation of the glucocorticoid receptor (GR). Furthermore, microRNA (miRNA) expression profiling has shown that Rg1 can modulate the expression of a subset of miRNAs to induce angiogenesis. Moreover, Rb1 was shown to be antiangiogenic through activation of a different pathway. These studies highlight the important functions of miRNAs on ginseng-regulated physiological processes. The aim of this study was to determine the angiogenic properties of Korean Red Ginseng extract (KGE). Methods and Results Combining in vitro and in vivo data, KGE at 500 μg/mL was found to induce angiogenesis. According to the miRNA sequencing, 484 differentially expressed miRNAs were found to be affected by KGE. Among them, angiogenic-related miRNAs; miR-15b, -23a, -214, and -377 were suppressed by KGE. Meanwhile, their corresponding angiogenic proteins were stimulated, including vascular endothelial growth factor, vascular endothelial growth factor receptor-2, endothelial nitric oxide synthase, and MET transmembrane tyrosine kinase. The miRNAs-regulated signaling pathways of KGE were then found by Cignal 45-Pathway Reporter Array, proving that KGE could activate GR. Conclusion KGE was found capable of inducing angiogenesis both in vivo and in vitro models through activating GR. This study provides a valuable insight into the angiogenic mechanisms depicted by KGE in relation to specific miRNAs.

The Wnt signaling pathway is known to serve an important role in the control of cell migration. The present study analyzed the mechanisms underlying the in vitro modulation of the migration of nasopharyngeal carcinoma (NPC) cells by the CREB‑binding protein/catenin antagonist and Wnt modulator ICG‑001. The results revealed that ICG‑001‑mediated inhibition of tumor cell migration involved downregulated mRNA and protein expression of the Wnt target gene cluster of differentiation (CD)44. It was also demonstrated that ICG‑001 downregulated the expression of CD44, and this effect was accompanied by restored expression of microRNA (miRNA)‑150 in various NPC cell lines. Using a CD44 3'‑untranslated region luciferase reporter assay, miR‑150 was confirmed to be a novel CD44‑targeting miRNA, which could directly target CD44 and subsequently regulate the migration of NPC cells. The present study provides further insight into the inhibition of tumor cell migration through the modulation of miRNA expression by the Wnt modulator ICG‑001.

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.

Background: Ginsenoside-Rg3, the pharmacologically active component of red ginseng, has been found to inhibit tumor growth, invasion, metastasis, and angiogenesis in various cancer models. Previously, we found that 20(R)-ginsenoside-Rg3 (Rg3) could inhibit angiogenesis. Since microRNAs (miRNAs) have been shown to affect many biological processes, they might play an important role in ginsenoside-mediated angiomodulation. Methods: In this study, we examined the underlying mechanisms of Rg3-induced angiosuppression through modulating the miRNA expression. In the miRNA-expression profiling analysis, six miRNAs and three miRNAs were found to be up- or down-regulated in vascular-endothelial-growth-factor-induced human-umbilical-vein endothelial cells (HUVECs) after Rg3 treatment, respectively. Results: A computational prediction suggested that mature hsa-miR-520h (miR-520h) targets ephrin receptor (Eph) B2 and EphB4, and hence, affecting angiogenesis. The up-regulation of miR-520h after Rg3 treatment was validated by quantitative real-time polymerase chain reaction, while the protein expressions of EphB2 and EphB4 were found to decrease, respectively. The mimics and inhibitors of miR-520h were transfected into HUVECs and injected into zebra-fish embryos. The results showed that overexpression of miR-520h could significantly suppress the EphB2 and EphB4 protein expression, proliferation, and tubulogenesis of HUVECs, and the subintestinal-vessel formation of the zebra fish. Conclusion: These results might provide further information on the mechanism of Rg3-induced angiosuppression and the involvement of miRNAs in angiogenesis.

Exosomes, a group of secreted extracellular nanovesicles containing genetic materials and signaling molecules, play a critical role in intercellular communication. During tumorigenesis, exosomes have been demonstrated to promote tumor angiogenesis and metastasis while their biological functions in nasopharyngeal carcinoma (NPC) are poorly understood. In this study, we focused on the role of NPC-derived exosomes on angiogenesis. Exosomes derived from the NPC C666-1 cells and immortalized nasopharyngeal epithelial cells (NP69 and NP460) were isolated using ultracentrifugation. The molecular profile and biophysical characteristics of exosomes were verified by Western blotting, sucrose density gradient and electron microscopy. We showed that the C666-1 exosomes (10 and 20 μg/ml) could significantly increase the tubulogenesis, migration and invasion of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. Subsequently, an iTRAQ-based quantitative proteomics was used to identify the differentially expressed proteins in C666-1 exosomes. Among the 640 identified proteins, 51 and 89 proteins were considered as up- and down-regulated (≥ 1.5-fold variations) in C666-1 exosomes compared to the normal counterparts, respectively. As expected, pro-angiogenic proteins including intercellular adhesion molecule-1 (ICAM-1) and CD44 variant isoform 5 (CD44v5) are among the up-regulated proteins, whereas angio-suppressive protein, thrombospondin-1 (TSP-1) was down-regulated in C666-1 exosomes. Further confocal microscopic study and Western blotting clearly demonstrated that the alteration of ICAM-1 and TSP-1 expressions in recipient HUVECs are due to internalization of exosomes. Taken together, these data strongly indicated the critical roles of identified angiogenic proteins in the involvement of exosomes-induced angiogenesis, which could potentially be developed as therapeutic targets in future.

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.

Ginsenoside Rb1 (Rb1) is the most predominant ginsenoside isolated from the roots of ginseng (Panax ginseng C. A. Meyer). This compound is active in various human biological pathways that are involved in human collagen synthesis and inhibition of cell apoptosis. In this study, the skin-whitening effects of Rb1 were investigated in B16 melanoma cells. Our results showed that Rb1 inhibited melanogenesis in α-melanocyte-stimulating hormone (α-MSH)-stimulated B16 cells in a dose-dependent manner, which collectively indicated that Rb1 may have skin-whitening effects and may be formulated into skin-whitening products for skin care. Accordingly, a ginsenoside collagen transdermal patch was developed as a vehicle to topically deliver Rb1 into pig skin. The percutaneous permeation, retention within skin, and release in vitro of Rb1 from seven transdermal patch formulas were studied. It was determined that the best formula for ginsenoside collagen transdermal patch is made of protein collagen hydrolysate powder (PCHP) 2.0% (w/w), methyl cellulose (MC) 0.5% (w/w), polyethyleneglycol 6000 (PEG6000) 0.5% (w/w), ginsenoside 0.036% (w/w), azone 0.4% (v/w), menthol 0.20% (w/w), and water.

Human disease caused by highly pathogenic avian influenza A (HPAI) (H5N1) is associated with fulminant viral pneumonia and mortality rates in excess of 60%. Acute respiratory syndrome (ARDS) has been found to be the most severe form of acute lung injury caused by H5N1 virus infection while cytokine dysregulation and viral replication are thought to contribute to its pathogenesis. In this study, the antiviral and anti-inflammatory effects of two indirubin derivatives: indirubin-3′-oxime (IM) and E804 on primary human peripherial blood-derived macrophages and type-I like pneumocytes (human alveolar epithelial cells) during influenza A (H5N1) virus infection were investigated. We found that both of the indirubin derivatives strongly suppress the pro-inflammatory cytokines including IP-10 (CXCL10), one of the key factors which contribute to the lung inflammation during H5N1 virus infection. In addition, we also demonstrated that the indirubin derivative delays the virus replication in the primary cell culture models. Our results showed that indirubin derivatives have a potential to be used as an adjunct to antiviral therapy for the treatment of severe human H5N1 disease.

Ginsenoside-Rg1 (Rg1) has been identified as potent proangiogenic agent, which plays an important role in wound healing promotion or treatment of ischemic injury. We previously reported that miR-214/eNOS pathway was involved in Rg1-induced angiogenesis. Following the same microRNA microarray profiling data, we proposed miR-15b would be another microRNA candidate involved in Rg1-induced angiogenesis. Using human umbilical vein endothelial cells (HUVECs), it was showed that Rg1 could reduce miR-15b expression rapidly and steadily, leading to a temporal induction of vascular endothelial growth factor receptor-2 (VEGFR-2). The in vitro motility and tubulogenesis via VEGFR-2 in Rg1-treated HUVECs were also demonstrated. Besides, the reduction of VEGFR-2 3'-UTR reporter activity by miR-15b in the luciferase reporter gene assay clearly indicated that miR-15b could affect the VEGFR-2 transcript through targeting its 3'-UTR region. Diminishing expression of endogenous miR-15b could increase VEGFR-2 expression and HUVECs migration and tubulogenesis; while over-expression of miR-15b was found to associate with the reduction of VEGFR-2 expression as well as cellular migration and tubulogenesis. In vivo, artificial increment of miR-15b by injecting Pre-miR-15b precursor into zebrafish embryos was also found to significantly suppress the subintestinal vessels formation. In conclusion, our results further demonstrated the involvement of microRNAs in Rg1-induced angiogenesis.

Rhizomes of Dioscorea species are traditionally used for relieving menopausal syndromes in Chinese medicine. The estrogen-stimulating bioactive principles have been demonstrated in our previous study. In this study, the estrogen-stimulating effects of proteins isolated from four Dioscorea species [D. alata L. (DA), D. zingiberensis C.H. Wright (DH), D. collettii var. hypoglauca (Palib.) S.J. PeiC.T. Ting (DH), and D. oppositifolia L. (DO)] have been investigated and compared. Microscopic authentication of four Dioscorea species was performed by using paraffin and powder sections of the rhizomes. The potential bioactive proteins of four Dioscorea species have been rapidly isolated by using a DOI-antibody affinity column chromatography on immobilized antibodies against on estradiol-stimulating protein from DO (DOI), and their bioactivity has been rapidly confirmed and compared by phenotypic (i.e., estradiol-stimulating effect) and target-based (i.e., STAR, aromatase, estrogen receptors) screening approaches. The estrogen-stimulating activity of bioactive proteins from DO is the highest. In addition, bioactive proteins from DO upregulated the estradiol-metabolizing enzymes (aromatase and steroidogenic acute regulatory protein). Meanwhile, bioactive proteins from DA, DH and DO upregulated estrogen receptor β (ERβ). All bioactive proteins did not change the expression of estrogen receptor β (ERα). The estrogen-stimulating bioactive proteins isolated from DO increased biosynthesis of estradiol and upregulated the protein expression of aromatase, steroidogenic acute regulatory protein, and ERβ. The results scientifically support the traditional use of DO in Chinese medicine for relieving menopausal syndrome. Besides, proteins from DA and DZ could also upregulate the translational levels of ERβ, and potentially reducing the risk of ovarian cancer, which also support the clinical use of them for treating female aging disorder. Graphical Abstract Comparative Analysis of DOI-like Proteins with Stimulating Activity on Ovarian Estradiol Biosynthesis from Four Different Dioscorea Species in vitro.

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-β 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., published_or_final_version

Wrinkle formation is one of the primary characteristics of skin aging, the major cause of wrinkle is the loss of structural protein type I collagen in dermal layer of skin. Topical application of natural substances to reduce wrinkle is gaining attention in recent years. Although a number of polyphenoic compounds are suggested to prevent ultraviolet-induced wrinkle, very few of them are able to increase type I collagen synthesis directly. Ginseng has been known in folk medicine of its beneficial effect to skin. The present study investigate the effect of ginsenoside on type I collagen induction in human dermal fibroblasts. Ginsenoside Rb₁ was shown to induce type I collagen expression in dermal fibroblasts in a dose- and time-dependent manner. Recent studies suggest the important post-transcriptional regulatory role of microRNAs; here we demonstrated that miR-25 can directly inhibit type I collagen protein expression, and treatment of fibroblasts with Rb₁ can reduce the inhibition by decreasing miR-25 level. Furthermore, we identified that the nuclear receptor, peroxisome proliferator-activated receptor-delta (PPARδ) is the key mediator of Rb₁-induced type I collagen expression. Knockdown of PPARδ by small-interference RNA abolished the Rb₁-induced type I collagen production and reversed the Rb₁-suppressed miR-25 expression. These results demonstrated that ginsenoside Rb₁ can increase target gene expression through transcriptional pathway, at the same time, inhibit the corresponding miRNA expression to minimize the translation repression. Furthermore, this study provide solid support of ginsenoside Rb₁-induced type I collagen expression, which warrant further study in the dermatological application of ginsenosides in skin disorders.

Ginsenosides are considered the major constituents that are responsible for most of the pharmacological actions of ginseng. However, some ginsenosides exist as stereoisomeric pairs, detailed and molecular exposition based on the structural differences of ginsenoside stereoisomers has not been emphasized in most studies. Here we explore the functional differences of ginsenoside Rg₃ stereoisomers on angiogenesis. In this study, we demonstrated the distinctive differential angiogenic activities of 20(S)-Rg₃ and 20(R)-Rg₃ stereoisomers. 20(S)-Rg₃ at micromolar concentration promotes human endothelial cells proliferation, migration and tube formation in vitro, as well as ex vivo endothelial sprouting. The effects induced by 20(S)-Rg₃ are significantly more potent than 20(R)-Rg₃. These effects are partially mediated through the activation of AKT/ERK-eNOS signaling pathways. Moreover, knockdown of peroxisome proliferator-activated receptor-gamma (PPARγ) by specific small interference RNA abolished the 20(S)-Rg₃-induced angiogenesis, indicating that PPARγ is responsible for mediating the angiogenic activity of Rg₃. Using reporter gene assay, the PPARγ agonist activity of 20(S)-Rg₃ has been found 10-fold higher than that of 20(R)-Rg₃. Computer modeling also revealed the differential binding is due to the chiral center of 20(S)-Rg₃ can form a critical hydrogen bond with Tyr473 of PPARγ ligand binding domain. The present study elucidated the differential angiogenic effects of Rg₃ stereoisomers by acting as agonist of PPARγ. The results shed light on the structural difference between two ginsenoside stereoisomers that can lead to significant differential physiological outcomes which should be carefully considered in the future development of ginsenoside-based therapeutics.

2-methoxyestradiol (2ME2), an endogenous metabolite of 17-β-estradiol, has been shown to induce apoptosis and cell cycle arrest in various tumor models. We have previously shown that 2ME2 induced endoreduplication in a well-differentiated nasopharyngeal carcinoma (NPC) HK-1 and a poorly differentiated C666-1 cell line. In the present study, we studied the survival factors involved in 2ME2-induced endoreduplicating NPC cells. In the HK-1 cells, knockdown of BcL-xL expression by siRNA resulted in the reduction of endoreduplication and an increase in the percentage of apoptosis. Further mechanistic study revealed that 2ME2 enhanced the expression of the phosphorylated form of STAT5 (p-STAT5-Y694), but not p-STAT3 (Y705) and p-STAT3 (S727), in the nucleus of HK-1 cells. Pre-treatment of cells with JAK/STAT inhibitor AG490 and STAT5 inhibitor resulted not only in the reduced expression of Bcl-xL, but also reduced the percentage of endoreduplicating cells. In contrast, 2ME2 enhanced the expression of p-STAT3 in the poorly differentiated C666-1 cells. Pharmacological inhibition of STAT3 or Bcl-2/xL resulted in a decrease in endoreduplication of C666-1 cells. Taken together, the expression of p-STAT5 and p-STAT3 was upregulated in 2ME2-induced endoreduplicating HK-1 and C666-1 cells, respectively. Combination of 2ME2 with Bcl-2/xL inhibitor is a novel strategy to reduce the formation of endoreduplicating cells during chemotherapeutic treatment of NPC. © 2011 Wiley Periodicals, Inc.

Ethnopharmacological relevance Panax ginseng is a precious traditional Chinese herbal medicine which has been utilized as herbal tonic for improving immunity. The active component, ginsenosides have been shown to possess various pharmacological functions including immunomodulation and cardiovascular protection. Aim of the study To investigate the immunomodulatory effect and anti-apoptotic effect of ginsenosides on avian influenza-infected human endothelial cells, and to present evidence for the cardiovascular protection by ginseng during influenza infection. Materials and methods Human umbilical vein endothelial cells (HUVECs) were infected with avian influenza H9N2/G1 to induce IP-10 production and cell death, cells were then incubated with ginsenosides PPT and Re. The level of IP-10 and microRNA was determined by ELISA and real-time PCR respectively. Cell death was determined by MTT, TUNEL and flow cytometry. Results Ginsenoside metabolite protopanaxatriol showed significant suppression effect on IP-10 production upon H9N2/G1 infection through up-regulation of miR-15b expression. In addition, ginsenoside-induced cytoprotection was reflected in the increase of cell viability. Data from flow cytometry analysis and TUNEL assay also showed that ginsenoside Re could protect ECs from H9N2/G1-induced apoptosis and DNA damage. Conclusions This report further supports the traditional belief for immunomodulatory effects of ginseng, also demonstrated the partial protective mechanism of ginsenosides on avian influenza infection and its related endothelial dysfunction.

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.

By appending a pair of carboxamidoquinoline pendants onto 1,2-diaminocyclohexane scaffold via N -alkylation, multifunctionalized ACAQ was designed and synthesized as a water soluble fluorescent ratiometric chemosensor for Zn 2+ . In 50% aqueous methanol buffer pH 7.4 solution, upon excitation at 316 nm, ACAQ (5 μM) displayed a selective ratiometric fluorescence changes with a shift from 410 to 490 nm in response to the interaction with Zn 2+ . After binding with 1 equiv of Zn 2+ , ACAQ exhibited a 12-fold enhancement in I 490 /I 410 characterized by a clear isoemissive point at 440 nm. The metal sensor binding mode was established by Job’s plot and the combined fluorescence and 1 H NMR spectroscopic method. The selectivity of the probe toward biological relevant cations and transition metal ions was proven to be good. In addition, the interference caused by Cu 2+ and Cd 2+ in the quantitation of Zn 2+ can be completely eliminated by the use of diethyldithiocarbamate as the screening agent. Exploitation of ACAQ as the sensing probe, ratiometric determination of Zn 2+ with the limit of detection (LOD) at 28.3 nm can be realized. In addition, the unique responsive properties of the probe toward Fe 3+ and Zn 2+ were used to construct a fluorescent switch. The membrane permeability of ACAQ to living cells and bio-imaging of Zn 2+ were demonstrated.

MicroRNAs (miRNAs) express differently in normal and cancerous tissues and thus are regarded as potent cancer biomarkers for early diagnosis. However, the short length and low abundance of miRNAs have brought challenges to the established detection assay in terms of sensitivity and selectivity. In this work, we present a novel miRNA detection assay in single-molecule level with total internal reflection fluorescence microscopy (TIRFM). It is a solution-based hybridization detection system that does not require pretreatment steps such as sample enrichment or signal amplification. The hsa-miR-21 (miR-21) is chosen as target miRNA for its significant elevated content in a variety of cancers as reported previously. Herein, probes of complementary single-stranded oligonucleotide were hybridized in solution to miR-21 and labeled with fluorescent dye YOYO-1. The fluorescent hybrids were imaged by an electron-multiplying charge-coupled device (EMCCD) coupled TIRFM system and quantified by single-molecule counting. This single molecule detection (SMD) assay shows a good correlation between the number of molecules detected and the factual concentration of miRNA. The detection assay is applied to quantify the miR-21 in extracted total RNA samples of cancerous MCF-7 cells, HepG2 cells, and normal HUVEC cells, respectively. The results agreed very well with those from the prevalent real-time polymerase chain reaction (qRT-PCR) analysis. This assay is of high potential for applications in miRNA expression profiling and early cancer diagnosis.

Photodynamic therapy (PDT) with a recently developed photosensitizer Zn-BC-AM was found to effectively induce apoptosis in a well-differentiated nasopharyngeal carcinoma (NPC) HK-1 cell line. Sustained activation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase (JNK) as well as a transient increase in activation of extracellular signal-regulated kinase (ERK) were observed immediately after Zn-BC-AM PDT. A commonly used p38 MAPK/JNK pharmacological inhibitor PD169316 was found to reduce PDT-induced apoptosis of HK-1 cells. PD169316 also prevented the loss of Bcl-2 and Bcl-xL in PDT-treated HK-1 cells. However, inhibition of JNK with SP600125 had no effect on Zn-BC-AM PDT-induced apoptosis while inhibition of ERK with PD98059 or p38 MAPK with SB203580 significantly increased Zn-BC-AM PDT-induced apoptosis. Further study showed that knockdown of the p38beta isoform with siRNA also increased Zn-BC-AM PDT-induced apoptosis, indicating that the anti-apoptotic effect of PD169316 in PDT-treated HK-1 cells was probably independent of p38 MAPK or JNK activation. Taken together, the results suggest that inhibition of p38beta and ERK may enhance the therapeutic efficacy of Zn-BC-AM PDT on NPC cells. It should be noted that data only based on the use of PD169316 should be interpreted in caution.

We describe a new nuclear staining strategy by in vivo anchoring Hoechst 33342, a commonly used nuclear staining organic dye, onto water-soluble N-acetylcysteine (NAC)-capped CdTe quantum dots (QDs). The strong H-bonding and electrostatic interaction between the organic dye and the QDs entice the positively charged nuclear staining agent in carrying effectively the bright QDs into the nucleus of living cells. Dynamic confocal imaging studies indicated that all visible light emitting NAC-capped QDs with emitting wavelength ranging from 530 to 602 nm can be successfully targeted to the nucleus of the living cells within 30 min. The minimum quantity of Hoechst 33342 as the anchoring agent required in carrying QDs to the nucleus is found to be 5 μg/mL. In essence, a simple, fast, and specific bioimaging of the nucleus in living cells by QDs is accomplished.

While semiconductor quantum dots produce little singlet oxygen, they may undergo Type I photoreactions to produce other reactive oxygen species (ROS) to kill cells. CdTe quantum dots coated with thioglycolic acid were used to test that possibility. Some thiol ligands were purposely removed to regenerate the surface electron traps that were passivated by the ligand. This allowed photoinduced electrons to dwell on the surface long enough to be gathered by nearby oxygen molecules to produce ROS. The photocytotoxicity of these quantum dots was tested on nasopharyngeal carcinoma cells. Photokilling was shown to be drug and light dose dependent. Using 0.6 mum quantum dots for incubation and 4.8 J cm(-2) for irradiation, about 80% of the cells were annihilated. These quantum dots promised to be potent sensitizers for photoannihilation of cancer cells.

Ginsenosides, the active components of the famous Chinese herb ginseng, have been suggested to possess cardiovascular-protective effects. The mechanism of ginsenosides is believed to be associated with their ability to prevent cellular oxidative stress. The purpose of this study was to explore the cytoprotective effects of the ginsenoside protopanaxatriol (PPT) on hydrogen peroxide (H(2)O(2))-induced endothelial cell injury and cell death. Pretreatment of human umbilical vein endothelial cells (HUVECs) with PPT for 24 h was able to protect the cells against H(2)O(2)-induced injury. In addition to cell death, pretreatment with PPT could also reduce H(2)O(2)-induced DNA damage, overactivation of the DNA repair enzyme PARP-1, and concomitant depletion of the intracellular substrate NAD(+). Furthermore, PPT could reverse the decrease in ATP/ADP ratio caused by H(2)O(2). The metabolism of glutathione was also changed. H(2)O(2) could induce a significant decrease in GSH level resulting in a decrease in the GSH/GSSG ratio. This could be prevented by pretreatment with PPT. The action was associated with increasing activities of the GSH-metabolizing enzymes glutathione reductase and glutathione peroxidase. These findings suggest that the ginsenoside PPT could protect HUVECs against H(2)O(2)-induced cell death via its action against oxidative stress, which may be responsible for the cardiovascular-protective action of ginseng.

An amphiphilic porphyrin appended with a Ru(II)–polypyridyl complex (Ru–P) showing a moderate two-photon absorption cross-section (178.0 ± 26.8 GM), 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.

Epidermal growth factor receptor (EGFR), a receptor often expressed in nasopharyngeal carcinoma (NPC) cells, is one of the recently identified molecular targets in cancer treatment. In the present study, the effects of combined treatment of Zn-BC-AM PDT with an EGFR inhibitor AG1478 were investigated. Well-differentiated NPC HK-1 cells were subjected to PDT with 1 microM of Zn-BC-AM and were irradiated at a light dose of 1 J/cm(2) in the presence or absence of EGFR inhibitor AG1478. Specific protein kinase inhibitors of downstream EGFR targets were also used in the investigation. EGFR, Akt, and ERK were found constitutively activated in HK-1 cells and the activities could be inhibited by the EGFR inhibitor AG1478. A sub-lethal concentration of AG1478 was found to further enhance the irreversible cell damage induced by Zn-BC-AM PDT in HK-1 cells. Pre-incubation of the cells with specific inhibitors of EGFR (AG1478), PI3k/Akt (LY294002), or MEK/ERK (PD98059) before light irradiation were found to enhance Zn-BC-AM PDT-induced formation of apoptotic cells. The efficacy of Zn-BC-AM PDT can be increased through the inhibition of EGFR/PI3K/Akt and EGFR/MEK/ERK signaling pathways in NPC cells. Combination therapy with Zn-BC-AM PDT and EGFR inhibitors may further be developed for the treatment of advanced NPC.

The present study aims to determine the role of mitogen-activated protein kinases (MAPKs) in hypericin-mediated photodynamic therapy (HY-PDT)-induced apoptosis of the HK-1 nasopharyngeal carcinoma (NPC) cells. HY-PDT was found to induce proteolytic cleavage of procaspase-9 and -3 in HK-1 cells. Apoptotic nuclei were observed at 6 h after PDT whereas B-cell leukemia/lymphoma-2-associated-X-protein (Bax) translocation and formation of Bax channel is responsible for the cell death. Increase in phosphorylation of p38 MAPKs and c-Jun N-terminal kinase 1/2 (JNK1/2) was detected at 15-30 min after HY-PDT. The appearance of phosphorylated form of p38 MAPKs and JNK1/2 was inhibited by the singlet oxygen scavenger l-histidine. HY-PDT-induced cell death was enhanced by the chemical inhibitors for p38 MAPKs (SB202190 and SB203580), but not by the JNKs inhibitor SP600125. Knockdown of the p38alpha and p38beta MAPK isoforms by small interfering RNA (siRNA) are more effective than the p38delta in enhancing PDT-induced cell death. Augmentation of apoptosis by p38alpha or p38beta knockdown is also correlated with the increased proteolytic cleavage of procaspase-9 after HY-PDT treatment. Our results suggested that HY-PDT activated p38 MAPKs through the production of singlet oxygen. Inhibition of p38 MAPKs with chemical inhibitors or siRNA enhances HY-PDT-induced apoptosis of the HK-1 NPC cells.

Background and purpose: Ginsenosides are used widely for medicinal purposes, but the mechanisms of their action are still unclear, although there is some evidence that these effects are mediated by nuclear receptors. Here we examined whether two metabolites of ginsenoside, protopanaxadiol (g-PPD) and protopanaxatriol (g-PPT), could modulate endothelial cell functions through the glucocorticoid receptor (GR) and oestrogen receptor (ER). Experiment approaches: The effects of g-PPD and g-PPT on intracellular calcium ion concentration ([Ca2+]i) and nitric oxide (NO) production in human umbilical vein endothelial cells (HUVECs) were measured using Fura-2-acetoxymethyl ester, 4-amino-5-methylamino-2′,7′-difluorofluorescein and Griess reagent. Effects on expression of GR and ER isoforms in HUVECs were determined using reverse transcriptase-/real-time PCR and immunocytochemistry. Phosphorylation of endothelial NO synthase (eNOS) was assessed by Western blotting. Results: Ginsenoside protopanaxadiol and g-PPT increased [Ca2+]i, eNOS phosphorylation and NO production in HUVECs, which were inhibited by the GR antagonist, RU486, the ER antagonist, ICI 182,780 and siRNA targeting GR or ERβ. The NO production was Ca2+-dependent and the [Ca2+]i elevation in HUVECs resulted from both intracellular Ca2+ release and extracellular Ca2+ influx. Conclusions and implications: Ginsenoside protopanaxadiol and g-PPT were functional ligands for both GR and ERβ, through which these ginsenoside metabolites exerted rapid, non-genomic effects on endothelial cells. Mandarin translation of abstract

A novel ribosome-inactivating protein (RIP) designated alpha-kirilowin was isolated from the seeds of Trichosanthes kirilowii. The molecular weight of alpha-kirilowin was estimated by SDS-polyacrylamide gel electrophoresis to be 28 800 Da, which is slightly larger than another previously characterized ribosome-inactivating protein, beta-kirilowin. The amino-acid composition of alpha-kirilowin grossly resembled beta-kirilowin and other ribosome-inactivating proteins isolated from T. kirilowii tissues, including trichokirin, trichosanthin and karasurin. Intense immunological cross-reactivity between the two kirilowins was detected by immunodiffusion. The N-terminal sequence of alpha-kirilowin was identical to that of beta-kirilowin, at least in the first ten residues. Peptide fingerprinting indicated both kirilowins were closely related. Biological activities as determined by inhibition of protein synthesis in a cell-free system, suppression of [3H]-thymidine incorporation into mouse melanoma cells and induction of abortion in mice were very similar for both kirilowins. We propose that the size difference between alpha- and beta-kirilowin is either due to a C-terminal extension in alpha-kirilowin or differences in glycosylation, or a combination of both.

We have investigated the neurite growth-stimulating properties of euxanthone, a xanthone derivative isolated from the Chinese medicinal plant Polygala caudata. Euxanthone was shown to exert a marked stimulatory action on neurite outgrowth from chick embryo dorsal root ganglia explanted in collagen gels, in the absence of added neurotrophins. It was also shown to promote cell survival in explanted chick embryo ganglia, and to stimulate neurite outgrowth from isolated adult rat primary sensory neurons in vitro. The further finding that euxanthone stimulates neurite outgrowth from explants of chick embryo retina and ventral spinal cord suggests an action on signaling pathways downstream of neuronal receptors for specific neurotrophic factors. Consistent with this, euxanthone did not promote neurite outgrowth from non-transfected PC12 cells, or from PC12 cells transfected with TrkB or TrkC, under conditions in which these cells extended neurites in response to, respectively, the neurotrophins nerve growth factor, brain-derived neurotrophic factor and neurotrophin 3. Western blot analysis of euxanthone-stimulated dorsal root ganglion explants showed that expression of phospho-mitogen-activated protein (MAP) kinase was up-regulated after 1 h of euxanthone-treatment. Inhibition of the MAP kinase pathway using PD98059, a specific inhibitor of MAP kinase kinase, blocked all euxanthone-stimulated neurite outgrowth. However, analysis of phospho-Akt expression indicated that the phosphatidylinositol-3 kinase-Akt pathway, another major signaling pathway engaged by neurotrophins, is not significantly activated by euxanthone. These results suggest that euxanthone promotes neurite outgrowth by selectively activating the MAP kinase pathway.

Background and purpose: 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. Experimental approaches: Rb1-induced PEDF was determined by real-time PCR and western blot analysis. The anti-angiogenic effects of Rb1 were demonstrated using endothelial cell tube formation assay. Competitive ligand-binding and reporter gene assays were employed to indicate the interaction between Rb1 and the oestrogen receptor (ER). Key results: Rb1 significantly increased the transcription, protein expression and secretion of PEDF. Targeted inhibition of PEDF completely prevented Rb1-induced inhibition of endothelial tube formation, suggesting that the anti-angiogenic effect of Rb1 was PEDF specific. Interestingly, the activation of PEDF occurred via a genomic pathway of ERβ. Competitive ligand-binding assays indicated that Rb1 is a specific agonist of ERβ, but not ERα. Rb1 effectively recruited transcriptional activators and activated an oestrogen-responsive reporter gene. Furthermore, Rb1-mediated PEDF activation and the subsequent inhibition of tube formation were blocked by the ER antagonist ICI 182,780 or transfection of ERβ siRNA, indicating ERβ dependence. Conclusions and implications: Here we show for the first time that the Rb1 suppressed the formation of endothelial tube-like structures through modulation of PEDF via ERβ. These findings demonstrate a novel mechanism of the action of this ginsenoside that may have value in anti-cancer and anti-angiogenesis therapy. British Journal of Pharmacology (2007) 152, 207–215; doi:10.1038/sj.bjp.0707359

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 Parkinson's 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.

Selenium-containing phycocyanin (Se–PC) and its different aggregates – monomer (αβ), trimer (αβ) 3 and hexamer (αβ) 6 , which are represented as Se–PC1, Se–PC2 and Se–PC3, as well as their α and β subunits, were prepared and purified from selenium-rich Spirulina platensis . The Se speciation in Se–PC, its aggregates and subunits, as well as the antioxidant activities of these compounds against free radicals of superoxide, hydrogen peroxide and 2,2-diphenyl-1-picryl-hydrazyl (DPPH ) were investigated. By gel filtration on SephadexG-200 column and SDS-PAGE, the molecular weight of Se–PC1, 2, and 3 were determined to be 38.5, 116 and 247 kDa, respectively, and these of α and β subunits were 17.8 and 19.5 kDa, respectively. Se concentrations of Se–PC1, 2 and 3 were of 197, 289 and 422 μg/g protein, respectively, and those of α and β subunits were 126 and 187 μg/g protein, respectively, which suggested that the Se content of Se–PC was decreased in the dissociation process of Se–PC and highly associated with its aggregation state. The antioxidant activities of Se–PC, its aggregates and subunits against different free radicals were found to be variable. Compared with phycocyanin without bound Se, although there were no differences among the DPPH -scavenging activities of Se–PC, its α and β subunits and the pure phycocyanin, the superoxide and hydrogen peroxide radical-scavenging activities of Se–PC, as well as Se–PC2 and Se–PC3, were significantly higher and proved to be positively correlated with their Se contents.

Therapeutic angiogenesis has been implicated in ischemic diseases and wound healing. Ginsenoside-Rg 1 (Rg 1 ), 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 Rg 1 -induced angiogenesis has not been fully explored. In this report, we found that stimulating endothelial cells with Rg 1 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-Rg 1 was found to increase MET protein expression in a time-dependent manner. We further demonstrated that ginsenoside-Rg 1 -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-Rg 1 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-Rg 1, and provided molecular evidence that warrant further development of natural compound as novel angiogenesis-promoting therapy.

Nasopharyngeal carcinoma (NPC) is an EBV-associated epithelial malignancy prevalent in southern China. Presence of treatment-resistant cancer stem cells (CSC) may associate with tumor relapse and metastasis in NPC. ICG-001 is a specific CBP/β-catenin antagonist that can block CBP/β-catenin-mediated transcription of stem cell associated genes and enhance p300/β-catenin-mediated transcription, thereby reducing the CSC-like population via forced differentiation. In this study, we aimed to evaluate the effect of ICG-001 on the CSC-like population, and the combination effect of ICG-001 with cisplatin in the C666-1 EBV-positive NPC cells. Results showed that ICG-001 inhibited C666-1 cell growth and reduced expression of CSC-associated proteins with altered expression of epithelial-mesenchymal transition (EMT) markers. ICG-001 also inhibited C666-1 tumor sphere formation, accompanied with reduced SOX2 hi /CD44 hi CSC-like population. ICG-001 was also found to restore the expression of a tumor suppressive microRNA-145 (miR-145). Ectopic expression of miR-145 effectively repressed SOX2 protein expression and inhibited tumor sphere formation. Combination of ICG-001 with cisplatin synergistically suppressed in vitro growth of C666-1 cells and significantly suppressed growth of NPC xenografts. These results suggested that therapeutically targeting of the CBP/β-catenin signaling pathway with ICG-001 can effectively reduce the CSC-like population and combination with cisplatin can effectively suppress the growth of NPC., link_to_OA_fulltext

The photodynamic properties of pyropheophorbide-a methyl ester (MPPa), a semi-synthetic photosensitizer derived from chlorophyll a, were evaluated in a human nasopharyngeal carcinoma HONE-1 cell line. MPPa was non-toxic to the HONE-1. At the concentrations of 0.5-2μM, MPPa-mediated a drug dose-dependent photocytotoxicity in the HONE-1 cells. Confocal microscopy revealed a subcellular localization of MPPa in mitochondria and the Golgi apparatus. MPPa PDT-induced apoptosis was associated with the collapse of mitochondrial membrane potential, release of cytochrome c, the up-regulation of endoplasmic reticulum (ER) stress proteins (calnexin, Grp 94 and Grp78), and the activation of caspases-3 and -9. The photocytotoxicity was reduced by the corresponding specific caspase inhibitors. MPPa PDT-treated HONE-1 cells also up-regulated the gene expression of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) and beta-chemokines (MIP-1β, MPIF-1, and MPIF-2). These results suggest that the MPPa may be developed as a chlorophyll-based photosensitizer for the treatment of nasopharyngeal carcinoma.

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 beta-catenin/T-cell factor-dependent pathway via the GR. Rg1 stimulation resulted in an increase in the level of beta-catenin, culminating its nuclear accumulation, and subsequent activation of VEGF expression. Transfection of a stable form of beta-catenin (S37A) or the use of a glycogen synthase kinase 3beta inhibitor to stabilize beta-catenin induced VEGF synthesis, whereas small interfering RNA-mediated down-regulation of beta-catenin did not, confirming that the effect was beta-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 3beta. In addition, the GR antagonist RU486 was able to inhibit Rg1-induced PI3K/Akt and beta-catenin activation. These findings provide new insights into the mechanism responsible for Rg1 functions.

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.

A novel microarray strategy, three-color cDNA microarray experiment, is originally applied to assess drug effects on gene expression patterns of “target disease.” By adding Alexa 594 as a dye-label for the third target sample, it is made possible to monitor changes in gene expression in response to disease and generate clues to gene function with therapeutic intervention only on one array. A new kind of graph, hexaMplot, is constructed to illustrate the meaningful expression patterns of normal-disease-drug in three-color experiment. The therapeutic and side effect of drugs can be understood and indicated by the different regions of hexaMplot. And the testing of correlation coefficient is explored as a statistical tool of the assessment. Such a methodology may prove useful in shortening the cycle, reducing the cost, and improving the efficiency of drug discovery and development.

Euxanthone, a neuritogenic agent isolated from the medicinal herb Polygala caudata, has been shown to induce morphological differentiation and neurite outgrowth in murine neuroblastoma Neuro 2a cells (BU-1 subclone). In order to elucidate the underlying mechanisms of euxanthone-induced neurite outgrowth, a proteomic approach was employed. In the present study, two dimensional (2-D) gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight (MALDI-ToF) mass spectrometry were performed to investigate the alterations in protein expression profile of euxanthone-treated BU-1 cells. Fourteen identified proteins were changed in expression levels after induction of neurite growth. These proteins included participants in transcription and cell cycle regulation, calcium influx and calcium signaling, fatty acid metabolism, cytoskeleton reorganization, casein kinase signal transduction, putative transbilayer amphipath transport and protein biosynthesis. Among the 14 identified proteins, E2F transcription factor 5 (E2F-5) was significantly up-regulated after euxanthone treatment. Go6976, a protein kinase C (PKC) α/βI inhibitor, was found to inhibit neuritogenesis and expression of E2F-5 in the euxanthone-treated BU-1 cells, while SH-6, the Akt/PKB inhibitor, had no inhibitory effect. The gene silencing of E2F-5 by small interfering RNA (siRNA) was found to abolish the euxanthone-induced neurite outgrowth. In conclusion, these results indicated that the transcription factor E2F-5 was actively involved in the regulation of euxanthone-induced neurite outgrowth via PKC pathway.

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-1beta 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.

High-speed counter-current chromatography was successfully applied for the first time to the isolation and purification of the bioactive carotenoid zeaxanthin from the cyanobacterium Microcystis aeruginosa. The crude zeaxanthin was obtained by extraction with organic solvents after the microalgal sample had been saponified. Preparative high-speed counter-current chromatography with a two-phase solvent system composed of n-hexane-ethyl acetate-ethanol-water (8:2:7:3, v/v/v/v) was successfully performed yielding zeaxanthin at 96.2% purity from 150 mg of the crude extract in a one-step separation. The recovery of zeaxanthin was 91.4%. This was also the first report that zeaxanthin was successfully separated and purified from microalgae. (C) 2004 Published by Elsevier B.V.

Metabolism of an anti-tumor active component of Panax ginseng, ginsenoside (20R)-Rg(3), was studied for better understanding its pharmacokinetics in rat. LC-MS was used to determine Rg(3) and its metabolites in rat plasma, urine and feces samples. An average half-life of 18.5 min was obtained after the ginsenoside was intravenously dosed at 5 mg/kg. However, Rg(3) was not detected in rat plasma collected after oral administration at 100 mg/kg. Only 0.97-1.15% Rg(3) of the dosed amount was determined in feces. Hydrolysis and oxygenated metabolites were detected and identified in feces collected after oral administration by using LC-MS and MS-MS.

The liver is the major organ for the metabolism of homocysteine (Hcy) and production of insulin-like growth factor 1 (IGF-1). Hcy metabolism and IGF-1 synthesis may be impaired in chronic liver diseases. The study investigated the regulatory effect of a Chinese herbal suppository, Vitalliver, on Hcy and IGF-1, as well as their relationship in patients with hepatitis B infection. Forty patients with chronic hepatitis B virus (HBV) infection without cirrhosis, 25 males and 15 females, were observed for changes in Hcy and IGF-1 after the administration of Vitalliver (one nightly) for a period of 3 months. Serum levels of Hcy, IGF-1 and IGFBP-3 were measured at baseline, and at 1 month and 3 months after treatment. Vitalliver reduced Hcy levels significantly (p = 0.001) from 9.7 +/- 2.8 to 9.0 +/- 2.1 micromol/L after treatment of 3 months. Furthermore, the IGF-1 levels increased significantly (p < 0.001) from 170.2 +/- 81.8 to 212.8 +/- 80.9 ng/mL at 1 month and 187.5 +/- 72.3 ng/mL at 3 months (p = 0.001) after treatment. In conclusion, it is speculated Vitalliver may have a self-regulatory effect on the release of IGF-1 in HBV patients without liver cirrhosis.

Si-Jun-Zi decoction (SJZD), a traditional Chinese herbal prescription, has been used clinically for treating patients with disorders of the digestive system. Previous studies indicated that the polysaccharides of SJZD (SJZPS) are the active components contributing towards its pharmacological effects in improving gastrointestinal function and immunity. However, the protective and restitutive effects on intestinal epithelial cells remain unknown. In the present study, SJZPS were first extracted and chemically characterized. Then their stimulatory and restitutive effects on intestinal epithelial cells (IEC-6 cells) were elicited by differentin vitromodels including migration of wounded IEC-6 cells and cell proliferation. Results indicated that SJZPS not only protects the cells against the harmful impairment of indomethacin but also enhances re-epithelialization of a wounded monolayer at an optimal dose of 100 μg/ml at 24 h incubation. To elucidate the modulatory effect of SJZPS on wounded IEC-6 cells at the molecular level, an oligonucleotide microarray was employed to study differential gene expression of SJZPS-treated IEC-6 cells and the candidate genes were validated by RT-PCR. There was increased expression of genes coding for ion channels and transporters, which are critical to cell migration and restoration of wounded intestinal cells, suggesting a possible mechanism for re-epithelialization. In conclusion, our data show for the first time that SJZPS can enhance intestinal restitution and protect against indomethacin-induced damage of intestinal epithelial cells. These findings provide new insight into the mechanism of action of a traditional Chinese herbal prescription, SJZD, in intestinal wound restitution.

In vivo metabolism and pharmacokinetic studies on rat were conducted for ginsenoside Rh2, one of the components from ginseng that shows promise of anticancer activity. Liquid chromatography/mass spectrometry (LC/MS) and tandem mass spectrometry (MS/MS) with electrospray ionization were used to determine Rh2 and its metabolites in rat plasma, urine and feces. An average half-life of 16 min in plasma was obtained after intravenous administration to male Sprague-Dawley rats at 5 mg/kg. No Rh2 was detected in plasma samples collected from 0 to 24 h following oral administration at 100 mg/kg, and only 0.12-0.25% of the dosed amount was found in the feces samples collected from 0 to 48 h after oral administration at 100 mg/kg. Three metabolites of Rh2 were detected in the feces samples. Oxygenation and deglycosylation were found to be the major metabolic pathways of Rh2. Intense metabolism, rather than excretion, appears to be the reason for the fast clearance of this ginsenoside.

Photodynamic therapy (PDT) is recently developed as an effective treatment for malignant disease. In PDT, the photosensitizer eradicates tumour by induction of apoptosis. In this study, we investigated the mechanistic actions of a recently developed second generation photosensitizer, Zn-BC-AM, on nasopharyngeal carcinoma (NPC) cells. Zn-BC-AM was found to localize in the mitochondria, endoplasmic reticulum (ER), and golgi body. Photoactivation of Zn-BC-AM loaded NPC cells resulted in a rapid collapse of mitochondrial membrane potential (Deltapsim) (15 min), followed by the release of cytochrome c (1 h), and activation of caspases-9 and -3 (4 h). Expression of ER chaperones Bip/Grp78 and Grp94, and ER resident lectin-like chaperone calnexin (CNX) was also enhanced in PDT-stressed NPC cells. Caspase-12, an important caspase involved in ER stress-induced apoptosis, was also activated. Inhibition of Ca2+ uptake into mitochondria by ruthenium red (RR) or loading the cells with EGTA-AM, an agent that buffers intracellular Ca2+ released from ER, resulted in a significant reduction of Zn-BC-AM PDT-induced cell death. These observations suggest that both ER and mitochondria are the subcellular targets of Zn-BC-AM. Effective activation of ER- and mitochondria-mediated apoptotic pathways is responsible for Zn-BC-AM PDT-induced NPC cell death.

Homocysteine (Hcy) is a by-product of methionine metabolism. An imbalance of Hcy in the body may lead to hyperhomocysteinemia, a condition with elevated Hcy concentration in blood that may be one of the risk factors responsible for the development of several vascular diseases (thromboembolism, atherosclerosis, stroke, vascular diseases and dementia). Radix Salvia miltiorrhiza (Danshen), a well-known Chinese medicinal herb that can activate and improve blood microcirculation, is noticeable for its beneficial effect in treating cardiovascular diseases. The present study is to demonstrate the protective effect of Danshen extract against the homocysteine-induced adverse effect on human umbilical vein endothelial cell (HUVEC). Homocysteine (5 mM) not only decreased the cell viability but also caused the disruption of capillary-like structure formation in vitro. The protective effect of Danshen aqueous extract and its active compounds on endothelial cell function were demonstrated through an in vitro tube formation assay, which mimics the new blood vessel formation. To identify the active components in the aqueous extract of Danshen, the content was characterized by instrumental analysis using high performance liquid chromatography with diode array detector (DAD) and electrospray tandem mass spectrometry (ESI-MS/MS). Interestingly, Danshen extract and its pure compounds showed different effectiveness in protecting HUVEC against Hcy-induced injury according to the following descending order: Danshen aqueous extract, 3-(3,4-dihydroxy-phenyl)-2-hydroxy-propionic acid (Danshensu), protocatechuic acid, catechin and protocatechualdehyde. We believed that such findings might provide evidence in understanding the beneficial effects of Danshen on the cardiovascular system.

Algal toxins have various adverse implications on the ecosystem directly and indirectly. Cyanotoxins, a group of algal toxins produced by toxic cyanobacteria, are of great concern. The understanding of their occurrence, properties, detection, and removal from water bodies is of importance for monitoring and management of cyanotoxins in freshwater bodies. Keywords: algal toxins; cyanotoxins; cyanobacteria; hepatotoxin-producing species; cyanobacterial bloom