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ABSTRACT: Natural products are some of the most important sources of lead compounds for drug discovery. The advanced isolation technique of lead compounds of natural origin using therapeutically relevant bioassays is capable of enhancing work efficiency from complex multiconstituent extracts. In the present study, a bioassay-guided isolation strategy combined with bioactivity screening was used to identify novel angiogenesis inhibitors from licorice (Glycyrrhiza uralensis Fisch.) based on the zebrafish model and rapid preparative separation by high-speed countercurrent chromatography. Zebrafish embryos at 24 h postfertilization were chosen as the angiogenesis inhibition model for bioactivity screening. A solvent system (n-hexane-ethyl acetate-methanol-water) with different ratios was optimized and applied in the high-speed countercurrent chromatography separation of two fractions, Fr5 and Fr6, from the ethyl acetate extract of licorice. Blood circulation and vascular outgrowth in intersegmental vessels were found to be simultaneously inhibited by isoliquiritigenin and isolicoflavonol in a dose-dependent manner. Thus, these two compounds were identified and considered as active inhibitors against angiogenesis. These experimental results indicate that zebrafish bioassays combined with high-speed countercurrent chromatography may provide an alternative pathway for the rapid isolation of bioactive natural products.
Journal of Separation Science 05/2012; 35(9):1167-72. · 2.73 Impact Factor
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ABSTRACT: A fundamental aspect of cancer development is cancer cell proliferation. Seeking for chemical agents that can interfere with
cancer cell growth has been of great interest over the years. In our study, we found that a benzoxazine derivative, (6-tert-butyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-3-yl)
methanol (TBM), could inhibit cell growth and caused significant cell cycle arrest in pulmonary adenocarcinoma A549 and H460
cells with wild-type p53, while not affecting the cell cycle distribution in p53-deleted H1299 lung adenocarcinoma cells. Since P53 plays an important role in regulating cell cycle progression, we analyzed
the protein level of p53 by Western blot, and detected a significant elevation of p53 level after TBM treatment in A549 and
H460 cells. The data suggested that TBM might specifically inhibit the proliferation of p53 wild-type lung adenocarcinoma
cells through a p53-dependent cell cycle control pathway. More interestingly, results indicated that TBM might serve as a
useful tool for studying the molecular mechanisms of lung cancer cell growth and cell cycle control, especially for the biologic
process regulated by P53.
Keywords(6-tert-butyl-3,4-dihydro-2H-benzo[b][1,4] oxazin-3-yl) methanol-lung adenocarcinoma cells-cell cycle arrest-p53
04/2012; 5(2):180-186.
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ABSTRACT: Emodin, a widely available herbal remedy, has a variety of pharmacological actions and valuable clinical applications. The potential effect of emodin on zebrafish (Danio rerio) embryos was evaluated. Zebrafish embryos were incubated with 0.1-2 μg/mL of emodin from 7 hours to 6 days postfertilization (dpf). Emodin, at concentrations of 0.25 μg/mL and above, negatively affected embryo survival and hatching success. Emodin induced a large suite of abnormalities on zebrafish embryos, such as edema, crooked trunk, and abnormal morphogenesis. To elucidate the mechanism of action, the transcript levels of drug-metabolism genes (CYP3A) and a multiple drug-resistance gene (MDR1) were detected by reverse-transcript polymerase chain reaction. Embryos showed increases in mRNA accumulation of CYP3A and MDR1. The above-described results indicated that emodin impaired zebrafish embryo development and some organ morphogenesis, and CYP3A and MDR1 were involved in the process. These findings suggest that emodin was toxic to zebrafish lavae at relatively low concentrations.
Drug and Chemical Toxicology 08/2011; 35(2):149-54. · 1.08 Impact Factor
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ABSTRACT: Celastrol is a terpenoid purified from Tripterygium wilfordii Hook F. As a natural product with pharmacological activities, this compound is a promising candidate for drug development. To provide more information about its toxicity for clinical trials, toxicity assessment of celastrol was conducted with zebrafish model in vivo. 1hour post-fertilization (hpf) embryos were treated with various concentrations of celastrol for 120h. Developmental phenotypes were observed and survival rates were recorded. The results showed that the hatching rates of embryos treated with 1.0μM or higher celastrol were significantly lower. Embryos exposed to 1.0μM celastrol had no blood flow in trunk vessels at 48hpf with a median effect concentration (EC(50)) of 0.94μM. At 72hpf serious edema in pericardial sac was observed in the surviving larvae (hatched from embryos treated with 1.5μM celastrol). Bent tails or hook-like tails were seen as 0.5μM celastrol and the EC(50) for tail malformation was 0.66 μM at 72hpf. The lethal effect of celastrol on zebrafish embryos was dose-dependent and the LC(50) values of celastrol on 1hpf embryos were approximately 1.40μM. These results indicate that celastrol affects the normal development of zebrafish embryo in μM concentrations.
Drug and Chemical Toxicology 10/2010; 34(1):61-5. · 1.08 Impact Factor
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ABSTRACT: Previously, we found that 6,8-dichloro-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine (DBO) promoted apoptosis of human umbilical vascular endothelial cells (HUVECs) deprived of growth factors. In this study, we aimed to investigate the effect of DBO and its mechanism of action on angiogenesis and apoptosis of HUVECs in the presence of fibroblast growth factor-2 (FGF-2), which promotes angiogenesis and inhibits apoptosis in vivo and in vitro. DBO significantly inhibited capillary-like tube formation by promoting apoptosis of HUVECs in the presence of FGF-2 in vitro. Furthermore, DBO elevated the levels of reactive oxygen species (ROS) and nitric oxide (NO) and increased the activity of NADPH oxidase and inducible nitric oxide synthase (iNOS) in promoting apoptosis under this condition. Moreover, when NADPH oxidase was inhibited by its specific inhibitor, dibenziodolium chloride (DPI), DBO could not elevate ROS and NO levels in HUVECs. The data suggest that DBO is a new modulator of apoptosis in vitro, and it might function by increasing the activity of NADPH oxidase and iNOS, subsequently elevating the levels of ROS and NO in HUVECs. The findings of this study provide a new small molecule for investigating the FGF-2/NADPH oxidase/iNOS signaling pathway in apoptosis.
Toxicology in Vitro 07/2009; 23(6):1039-46. · 2.78 Impact Factor
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ABSTRACT: Integrin beta4 is a tissue-specific protein, but its role in autophagy of lung adenocarcinoma cells is not clear. In this study, we used microtubule-associated protein 1 light chain 3 processing and acridine orange staining to reveal that knockdown of integrin beta4 by its specific siRNA induced autophagic cell death in A549 lung cancer cells. Next, we investigated the effects of siRNA-mediated downregulation of integrin beta4 on cell death and the level of p53. The proportion of dead cells and level of p53 were significantly increased. Inhibition of autophagy by the inhibitor 3-methyladenine attenuated the cell death induced by integrin beta4 knockdown. To further understand the relationship between p53 and integrin beta4 in autophagic cell death, we inhibited the expression of integrin beta4 by its specific siRNA in p53-mutated H322 lung cancer cells. Knockdown of integrin beta4 could not induce autophagic cell death in H322 cells. The data suggest that integrin beta4 is implicated in and associated with p53 in autophagy of lung cancer cells.
FEBS Journal 12/2008; 275(22):5725-32. · 3.79 Impact Factor
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ABSTRACT: Previously, we found that nine kinds of new morpholin-3-one derivatives could inhibit the growth of A549 lung cancer cells in a dose-dependent manner, but how they performed their function remained unknown. In this paper, we studied the effects of the three more effective morpholin-3-one derivatives {4-(4-chlorophenyl)-6-((4-nitrophenoxy) methyl) morpholin-3-one (1); 6-(4-chlorophenoxy)-4-(4-methoxyphenyl) morpholin-3-one (2); and 6-((4-nitrophenoxy) methyl)-4-phenylmorpholin-3-one (3)} on the cell cycle distribution, apoptosis, and the level of P53 and Fas that are two kinds of important proteins in the regulation of A549 cell growth and apoptosis. According to the results of cell viability, we selected 40 microg/ml of morpholin-3-one derivatives as the most appropriate concentration for the following study. The results showed that the morpholin-3-one derivatives partly blocked the cells at G1 phase, induced apoptosis, and elevated the level of P53 and Fas proteins significantly. The effect of the morpholin-3-one derivates was associated with translocation of P53 and clustering of Fas. Our data suggested that the morpholin-3-one derivates might be promising tools for elucidating the molecular mechanism of lung cancer cell apoptosis and they will be very potential candidates for developing anti-cancer drugs.
Bioorganic & Medicinal Chemistry 06/2007; 15(11):3889-95. · 2.92 Impact Factor
