Soluble E-cadherin promotes tumor angiogenesis and localizes to exosome surface.

The limitations of current anti-angiogenic therapies necessitate other targets with complimentary mechanisms. Here, we show for the first time that soluble E-cadherin (sE-cad) (an 80-kDa soluble form), which is highly expressed in the malignant ascites of ovarian cancer patients, is a potent inducer of angiogenesis. In addition to ectodomain shedding, we provide further evidence that sE-cad is abundantly released in the form of exosomes. Mechanistically, sE-cad-positive exosomes heterodimerize with VE-cadherin on endothelial cells and transduce a novel sequential activation of ß-catenin and NFκB signaling. In vivo and clinical data prove the relevance of sE-cad-positive exosomes for malignant ascites formation and widespread peritoneal dissemination. These data advance our understanding of the molecular regulation of angiogenesis in ovarian cancer and support the therapeutic potential of targeting sE-cad. The exosomal release of sE-cad, which represents a common route for externalization in ovarian cancer, could potentially be biomarkers for diagnosis and prognosis.

Angiosuppressive properties of marine-derived compounds-a mini review.

Angiogenesis, formation of new blood vessels from preexisting one, is a critical step of tumorgenesis of solid tumors. Therefore, antiangiogenic therapy is one of the promising approaches to control tumor growth. In the past 20 years, a lot of compounds have been tested for their antiangiogenic properties. Bevacizumab, Avastin®, the first antiangiogenic drug approved by the US FDA, has been widely used in clinic for treating cancer. Indeed, many synthetic compounds are highly toxic and exert side effects even though they are effective in inhibiting neovessel formation and cancer cell growth. Using natural compounds or their derivatives is one of the ways to solve these problems. Sinomenine and ginsenosides are common antiangiogenic and anticancer compounds that are extracted from herbal medicines. Recent findings suggested that marine algae-derived natural pigments also possess similar activities. It has been reported that fucoxanthin from Undaria pinnatifida, Siphonaxanthin from Codium fragile, can inhibit angiogenesis and cancer growth effectively. In conclusion, natural compounds derived from marine algae could provide a novel and safe source for new drug development in anticancer and antiangiogenic properties in the future.

Anti-inflammatory and antiviral effects of indirubin derivatives in influenza A (H5N1) virus infected primary human peripheral blood-derived macrophages and alveolar epithelial cells.

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.

MicroRNA-15b contributes to ginsenoside-Rg1-induced angiogenesis through increased expression of VEGFR-2.

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.

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

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

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 microm. Using this improved wounding device, the effects of epidermal growth factor and DL-alpha-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 angiosuppressive effects of 20(R)- ginsenoside Rg3.

Aberrant angiogenesis is an essential step for the progression of solid tumors. Thus anti-angiogenic therapy is one of the most promising approaches to control tumor growth. In this study, we examined the ability of 20(R)-ginsenoside Rg3 (Rg3), one of the active compounds present in ginseng root, to interfere with the various steps of angiogenesis. Rg3 was found to inhibit the proliferation of human umbilical vein endothelial cells (HUVEC) with an IC50 of 10 nM in Trypan blue exclusion assay. Rg3 (1-10(3) nM) also dose dependently suppressed the capillary tube formation of HUVEC on the Matrigel in the presence or absence of 20 ng/ml vascular endothelial growth factor (VEGF). The VEGF-induced chemoinvasion of HUVEC and ex vivo microvascular sprouting in rat aortic ring assay were both significantly attenuated by Rg3. In addition, Rg3 (150 and 600 nM) remarkably abolished the basic fibroblast growth factor (bFGF)-induced angiogenesis in an in vivo Matrigel plug assay. The Matrix metalloproteinases (MMPs), such as MMP-2 and MMP-9, which play an important role in the degradation of basement membrane in angiogenesis and tumor metastasis present in the culture supernatant of Rg3-treated aortic ring culture were found to decrease in their gelatinolytic activities. Taken together, these data underpin the anti-tumor property of Rg3 through its angiosuppressive activity.

Angiogenesis: from plants to blood vessels.

Angiogenesis is a major pathological component of diseases such as cancer and coronary heart disease. Although major advances have been made and encouraging clinical results obtained, safer and more effective approaches are required. The identification of new drugs from plants has a long and successful history, and certain proangiogenic and antiangiogenic plant components have been used in traditional Chinese medicine (TCM) for thousands of years. Similar to Western combination therapy, TCM uses mixtures of plant extracts, termed fufang, to maximize efficacy and minimize adverse effects or toxicity. More evidence-based research and chemical optimization of these compounds could further enhance the effectiveness of these plant-based medicines in angiotherapy.

Simultaneous detection of precore/basal core promoter mutations in hepatitis B virus using arrayed primer extension.

BACKGROUND: Hepatitis B is a major disease that causes serious public health problems worldwide. The loss of HBeAg expression due to point mutations or single nucleotide polymorphisms (SNPs) in the precore/basal core promoter region of the hepatitis B virus (HBV) is associated with hepatocellular cirrhosis and carcinoma. Simultaneous screening for these mutations is strongly advocated for monitoring disease development in HBV-infected patients. The aim of this study is to apply arrayed primer extension (APEX) for the detection of HBV SNPs at the precore/basal core promoter. METHODS AND RESULTS: We optimized APEX for simultaneous detection of eight potential sites of SNPs in the precore/basal core promoter region of HBV. The precore/basal core promoter regions of HBV from 36 HBV-infected patients were amplified by PCR. After purification and DNA fragmentation, the short, single-stranded HBV DNA fragments were allowed to hybridize with the oligonucleotides corresponding to the sites of SNPs immobilized on glass slides, followed by incorporation of different fluorescently labeled dideoxynucleotides. This allows fast and unequivocal discrimination between wild-type and mutant genotypes with high dideoxy-nucleotide incorporation efficiency, sensitivity, and specificity. The coexistence of both genotypes was also detected; this was undetected by DNA sequencing. CONCLUSION: The simultaneous detection of SNPs in HBV precore/basal core promoter by APEX enables large-scale diagnostic analysis, which can be extended to the whole HBV genome.

Effect of sinomenine on gene expression of the IL-1 beta-activated human synovial sarcoma.

Sinomenine is an alkaloid with pharmacological effects of anti-inflammation, anti-angiogenesis, anti-arthritis and immunosuppression. This study aimed to investigate the effect of sinomenine on gene expression of human synovial sarcoma cells (Hs701.T) activated by IL-1 beta. The proliferative effect of sinomenine was examined in the presence or absence of IL-1 beta by the [3H]-thymidine incorporation and MTT assay, respectively. Using DNA microarray technology and RT-PCR, the activating action of IL-1 beta and modulatory effect of sinomenine on Hs701.T were simultaneously determined. Results showed that IL-1 beta could stimulate the proliferation and gene expression of Hs701.T cells. Sinomenine could significantly inhibit proliferation of IL-1 beta-activated Hs701.T cells and suppress expression of 17 genes including IL-6, PlGF, Daxx, and HSP27. These genes were found to be important in tumor progression through the mediation of inflammation, cell adhesion, proliferation, apoptosis and angiogenesis. In conclusion, our study provides supplementary information for the further studies on the pharmacological effects of sinomenine acting on synovial sarcoma.

Elucidation of the mechanisms underlying the angiogenic effects of ginsenoside Rg(1) in vivo and in vitro.

The major active constituents of ginseng are ginsenosides, and Rg(1) is a predominant compound of the total extract. Recent studies have demonstrated that Rg(1) can promote angiogenesis in vivo and in vitro. In this study, we used a DNA microarray technology to elucidate the mechanisms of action of Rg(1). We report that Rg(1) induces the proliferation of HUVECs, monitored using [(3)H]-thymidine incorporation and Trypan blue exclusion assays. Furthermore, Rg(1) (150-600 nM) also showed an enhanced tube forming inducing effect on the HUVEC. Rg(1) was also demonstrated to promote angiogenesis in an in vivo Matrigel plug assay, and increase endothelial sprouting in the ex vivo rat aorta ring assay. Differential gene expression profile of HUVEC following treatment with Rg(1) revealed the expression of genes related to cell adhesion, migration and cytoskeleton, including RhoA, RhoB, IQGAP1, CALM2, Vav2 and LAMA4. Our results suggest that Rg(1) can promote angiogenesis in multiple models, and this effect is partly due to the modulation of genes that are involved in the cytoskeletal dynamics, cell-cell adhesion and migration.

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-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.

Effects of Si-Jun-Zi decoction polysaccharides on cell migration and gene expression in wounded rat intestinal epithelial cells.

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 different in vitro models 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 mug/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.

Identification of differentially expressed genes with multivariate outlier analysis.

DNA microarray offers a powerful and effective technology to monitor the changes in the gene expression levels for thousands of genes simultaneously. It is being widely applied to explore the quantitative alternation in gene regulation in response to a variety of aspects including diseases and exposure of toxicant. A common task in analyzing microarray data is to identify the differentially expressed genes under two different experimental conditions. Because of the large number of genes and small number of arrays, and higher signal-noise ratio in microarray data, many traditional approaches seem improper. In this paper, a multivariate mixture model is applied to model the expression level of replicated arrays, considering the differentially expressed genes as the outliers of the expression data. In order to detect the outliers of the multivariate mixture model, an effective and robust statistical method is first applied to microarray analysis. This method is based on the analysis of kurtosis coefficient (KC) of the projected multivariate data arising from a mixture model so as to identify the outliers. We utilize the multivariate KC algorithm to our microarray experiment with the control and toxic treatment. After the processing of data, the differential genes are successfully identified from 1824 genes on the UCLA M07 microarray chip. We also use the RT-PCR method and two robust statistical methods, minimum covariance determinant (MCD) and minimum volume ellipsoid (MVE), to verify the expression level of outlier genes identified by KC algorithm. We conclude that the robust multivariate tool is practical and effective for the detection of differentially expressed genes.