Kaempferol Identified by Zebrafish Assay and Fine Fractionations Strategy from Dysosma versipellis Inhibits Angiogenesis through VEGF and FGF Pathways.

Natural products are a rich resource for the discovery of therapeutic substances. By directly using 504 fine fractions from isolated traditional Chinese medicine plants, we performed a transgenic zebrafish based screen for anti-angiogenesis substances. One fraction, DYVE-D3, was found to inhibit the growth of intersegmental vessels in the zebrafish vasculature. Bioassay-guided isolation of DYVE-D3 indicates that the flavonoid kaempferol was the active substance. Kaempferol also inhibited the proliferation and migration of HUVECs in vitro. Furthermore, we found that kaempferol suppressed angiogenesis through inhibiting VEGFR2 expression, which can be enhanced by FGF inhibition. In summary, this study shows that the construction of fine fraction libraries allows efficient identification of active substances from natural products.

[Application of zebrafish models in drug screening].

Due to its small size, fast external development, transparent embryos, and amenability to genetic analysis, zebrafish has become an ideal vertebrate animal model. In addition to studies in genetics and developmental biology, zebrafish has also been widely used in human disease modeling and drug screening. As a small whole-organism model, zebrafish can be used to comprehensively test and evaluate the activity and side effect of a compound at the same time, fulfilling high content screening. Recently, new zebrafish disease models and screening technologies have been developed. A number of active compounds were identified and most of them have similar functions in mammal models. One compound prostaglandin E2 has been subjected to clinical trial to test if it can promote the growth of umbilical cord blood units after transplantation. Another compound leflunomide has also been approved in clinical trial to cure melanoma in combination with vemurafenib. These findings demonstrate that zebrafish model is appropriate for drug screening. This review summarizes the unique features of zebrafish model and the recent progresses of zebrafish based drug screening.

Genetic approach to evaluate specificity of small molecule drug candidates inhibiting PLK1 using zebrafish.

During the preclinical drug discovery process it remains a challenge to enable early elimination of candidate molecules that may have non-specific, off-target activities. Here, we use whole zebrafish embryo assays coupled with genetic analysis to address this issue. PLK1 (Polo-like kinase 1) is one of the key regulators that control mitotic entry, spindle assembly, chromosome segregation, and cytokinesis in the cell cycle. Since plk1 expression is abnormally up-regulated in several tumors, it is regarded as a good target for cancer therapy. A number of small-molecule inhibitors targeting PLK1 have been developed as reagents and anticancer drug candidates. It will be interesting to determine if these inhibitors indeed specifically target PLK1 in vivo. Bioinformatics analysis revealed that the zebrafish and human genomes share high homology across all PLK family members. In particular, PLK1 has a nearly identical 3-D structure between zebrafish and human. We selected three published PLK1 inhibitors, LFM-A13, ON01910, and thiazole-carboxamide 10A in our assay. When added at 2-cell stage, all of these inhibitors prevented embryos from dividing and caused cells to fuse into one large cell. When added at the later stage during zygotic mRNA transcription program initiation, embryos survived for 3 days but showed different phenotypes for each compound. Embryos treated with LFM-A13 appeared relatively normal. Embryos treated with ON01910 failed to properly develop trunk and tail regions while the head structure was unaffected. Embryos treated with thiazole-carboxamide 10A had a shorter body axis and deformed head structure. To determine which inhibitor is more selectively targeting PLK1, we inhibited PLK1 activity using anti-sense morpholino. Comparative analysis indicated that thiazole-carboxamide 10A could faithfully phenocopy zebrafish embryos genetically deficient of plk1. These findings demonstrate that these three PLK1 inhibitors, although well established by in vitro studies, have different off-target activities in vivo, and that thiazole-carboxamide 10A appears most specific to PLK1. Our studies suggest that zebrafish should be generally useful as an efficient in vivo model to evaluate specificity of small molecules designed to regulate any conserved target proteins through comparative analysis of genetic phenotypes.

[Preparation of magnetic solid liposome nanoparticles of paclitaxel].

AIM: To study a new way to prepare high-dosage paclitaxel entrapped magnetic targeted nanoparticles and evaluate its quality. METHODS: Fe3O4 nanoparticles are prepared by co-depositing, at the same time ultrasonic is used to decrease soft agglomerate of nanoparticles and increase disperse level of it. The property of nanoparticles surface is improved to make the integrating of liposome and nanoparticle to be tighter. At last, paclitaxel entrapped magnetic solid liposome nanoparticles have been prepared by microemulsion-curing under low-temperature. The loading efficiency and encapsulating rate were determined by reverse-phase high-perfomance chromatography. RESULTS: The nanoparticles have spherical shape. Diameter of nanoparticle ranged from 150 nm to 170 nm. 98.29% of the drug is entrapped in the particle. CONCLUSION: Magnetic susceptibility of nanoparticles is high, and the nanoparticles meet with the demand of targeted delivery system.