cAMP prevents TNF-induced apoptosis through inhibiting DISC complex formation in rat hepatocytes

The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide) assay is a classical method for screening cytotoxic anti-cancer agents. Candidate drugs from the MTT assay need in vivo models to test their efficiency and to assess the absorption, distribution, metabolism, excretion, and toxicity of the drugs. An in vivo screening model could increase the rate of development of anti-cancer drugs. Here, we used zebrafish to screen a library of 502 natural compounds and compared the results with those from an MTT assay of the MCF7 breast cancer cell line. We identified 59 toxic compounds in the zebrafish screen, 21 of which were also identified by the MTT assay, and 28 of which were already known for their anti-cancer and apoptosis-inducing effects. These compounds induced apoptosis and activated the p53 pathway in zebrafish within 3h treatment. Our results indicate that zebrafish is a simple, reliable and highly efficient in vivo tool for cancer drug screening, and could complement the MTT assay.     

Automated reporter quantification in vivo: high-throughput screening method for reporter-based assays in zebrafish

Reporter-based assays underlie many high-throughput screening (HTS) platforms, but most are limited to in vitro applications. Here, we report a simple whole-organism HTS method for quantifying changes in reporter intensity in individual zebrafish over time termed, Automated Reporter Quantification in vivo (ARQiv). ARQiv differs from current "high-content" (e.g., confocal imaging-based) whole-organism screening technologies by providing a purely quantitative data acquisition approach that affords marked improvements in throughput. ARQiv uses a fluorescence microplate reader with specific detection functionalities necessary for robust quantification of reporter signals in vivo. This approach is: 1) Rapid; achieving true HTS capacities (i.e., >50,000 units per day), 2) Reproducible; attaining HTS-compatible assay quality (i.e., Z'-factors of ≥0.5), and 3) Flexible; amenable to nearly any reporter-based assay in zebrafish embryos, larvae, or juveniles. ARQiv is used here to quantify changes in: 1) Cell number; loss and regeneration of two different fluorescently tagged cell types (pancreatic beta cells and rod photoreceptors), 2) Cell signaling; relative activity of a transgenic Notch-signaling reporter, and 3) Cell metabolism; accumulation of reactive oxygen species. In summary, ARQiv is a versatile and readily accessible approach facilitating evaluation of genetic and/or chemical manipulations in living zebrafish that complements current "high-content" whole-organism screening methods by providing a first-tier in vivo HTS drug discovery platform.     

Behavioral screening for neuroactive drugs in zebrafish

The larval zebrafish has emerged asa vertebrate model system amenable to small molecule screens for probing diverse biological pathways. Two large-scale small molecule screens examined the effects of thousands of drugs on larval zebrafish sleep/wake and photomotor response behaviors. Both screens identified hundreds of molecules that altered zebrafish behavior in distinct ways. The behavioral profiles induced by these small molecules enabled the clustering of compounds according to shared phenotypes. This approach identified regulators of sleep/wake behavior and revealed the biological targets for poorly characterized compounds. Behavioral screening for neuroactive small molecules in zebrafish is an attractive complement to in vitro screening efforts, because the complex interactions in the vertebrate brain can only be revealed in vivo.     

Zebrafish as a new animal model for movement disorders

The zebrafish, long recognized as a model organism for the analysis of basic developmental processes, is now also emerging as an alternative animal model for human diseases. This review will first provide an overview of the particular characteristics of zebrafish in general and their dopaminergic nervous system in particular. We will then summarize all work undertaken so far to establish zebrafish as a new animal model for movement disorders and will finally emphasize its particular strength – amenability to high throughput in vivo drug screening.     

Development and validation of an automated high-throughput system for zebrafish in vivo screenings

The zebrafish is a vertebrate model compatible with the paradigms of drug discovery. The small size and transparency of zebrafish embryos make them amenable for the automation necessary in high-throughput screenings. We have developed an automated high-throughput platform for in vivo chemical screenings on zebrafish embryos that includes automated methods for embryo dispensation, compound delivery, incubation, imaging and analysis of the results. At present, two different assays to detect cardiotoxic compounds and angiogenesis inhibitors can be automatically run in the platform, showing the versatility of the system. A validation of these two assays with known positive and negative compounds, as well as a screening for the detection of unknown anti-angiogenic compounds, have been successfully carried out in the system developed. We present a totally automated platform that allows for high-throughput screenings in a vertebrate organism.     

The Study of Glioma by Xenotransplantation in Zebrafish Early Life Stages

Zebrafish (Danio rerio) and their transparent embryos are becoming an increasingly popular tool for studying processes involved in tumor progression and in the search for novel tumor treatment approaches. The xenotransplantation of fluorescently labeled mammalian cancer cells into zebrafish embryos is an approach enabling relatively high-throughput in vivo analyses. The small size of the embryos as well as the relative simplicity of their manipulation and maintenance allow for large numbers of embryos to be processed efficiently in a short time and at low cost. Furthermore, the possibility of fluorescence microscopic imaging of tumor progression within zebrafish embryos and larvae holds unprecedented potential for the real-time visualization of these processes in vivo. This review presents the methodologies of xenotransplantation studies on zebrafish involving research on tumor invasion, proliferation, tumor-induced angiogenesis and screening for antitumor therapeutics. We further focus on the application of these zebrafish to the study of glioma; in particular, its most common and malignant form, glioblastoma.     

An investigation of the bioactivation potential and metabolism profile of Zebrafish versus human

The zebrafish model has been increasingly explored as an alternative model for toxicity screening of pharmaceutical drugs. However, little is understood about the bioactivation of drug to reactive metabolite and phase I and II metabolism of chemical in zebrafish as compared with human. The primary aim of our study was to establish the bioactivation potential of zebrafish using acetaminophen as a probe substrate. Our secondary aim was to perform metabolite profiling experiments on testosterone, a CYP3A probe substrate, in zebrafish and compare the metabolite profiles with that of human. The glutathione trapping assay of N-acetyl-p-benzoquinone imine demonstrated that zebrafish generates the same reactive metabolite as humans from the bioactivation of acetaminophen. Zebrafish possesses functional CYP3A4/5-like and UDP-glucuronosyltransferase metabolic activities on testosterone. Differential testosterone metabolism was observed among the two species. In silico docking studies suggested that the zebrafish CYP3A65 was responsible for the bioactivation of acetaminophen and phase I hydroxylation of testosterone. Our findings reinforce the need to further characterize the drug metabolism phenotype of zebrafish before the model can fully achieve its potential as an alternative toxicity screening model in drug research.     

Zebrafish bioassay-guided natural product discovery: isolation of angiogenesis inhibitors from East African medicinal plants

Natural products represent a significant reservoir of unexplored chemical diversity for early-stage drug discovery. The identification of lead compounds of natural origin would benefit from therapeutically relevant bioassays capable of facilitating the isolation of bioactive molecules from multi-constituent extracts. Towards this end, we developed an in vivo bioassay-guided isolation approach for natural product discovery that combines bioactivity screening in zebrafish embryos with rapid fractionation by analytical thin-layer chromatography (TLC) and initial structural elucidation by high-resolution electrospray mass spectrometry (HRESIMS). Bioactivity screening of East African medicinal plant extracts using fli-1:EGFP transgenic zebrafish embryos identified Oxygonum sinuatum and Plectranthus barbatus as inhibiting vascular development. Zebrafish bioassay-guided fractionation identified the active components of these plants as emodin, an inhibitor of the protein kinase CK2, and coleon A lactone, a rare abietane diterpenoid with no previously described bioactivity. Both emodin and coleon A lactone inhibited mammalian endothelial cell proliferation, migration, and tube formation in vitro, as well as angiogenesis in the chick chorioallantoic membrane (CAM) assay. These results suggest that the combination of zebrafish bioassays with analytical chromatography methods is an effective strategy for the rapid identification of bioactive natural products.     

Disruption of Ca2+-dependent cell-matrix adhesion enhances c-Src kinase activity, but causes dissociation of the c-Src/FAK complex and dephosphorylation of tyrosine-577 of FAK in carcinoma cells

The nonreceptor tyrosine kinase c-Src is activated in most invasive cancers. Activated c-Src binds to FAK in the focal adhesion complex, resulting in the activation of the c-Src/FAK signaling cascade, which regulates cytoskeletal functions. However, the mechanisms by which c-Src/FAK signaling is regulated during conditions of anchorage-independent growth, a hallmark of tumor progression, are not clearly known. Here, an in vivo approach to measure c-Src activity was studied using phospho-specific antibodies against phosphorylated Y418 of c-Src (Src[pY418]), an autophosphorylation site of c-Src, and phosphorylated Y577 of FAK (FAK[pY577]), a known substrate of c-Src. Using genetic and pharmacological approaches to modulate c-Src activity, we showed that the levels of Src[pY418] and FAK[pY577], and the formation of a c-Src/FAK[pY577] complex correlated with the activation state of c-Src in adherent cells. Interestingly, both the in vivo level of Src[pY418] and in vitro c-Src kinase activity were increased in carcinoma cells following disruption of Ca(2+)-dependent cell-matrix adhesion. In contrast, the level of FAK[pY577] and its association with c-Src were reduced in suspended cells. The amount of FAK[pY577] in suspended cells was recovered following attachment of rounded cells to fibronectin-coated polystyrene beads, indicating that cell spreading was not required for phosphorylation of FAK. Moreover, cells expressing activated c-Src showed sustained Src[Y418] phosphorylation, but required Ca(2+)-dependent cell adhesion for phosphorylation of FAK[Y577] and association of c-Src with FAK[pY577]. These findings indicate an important role of integrin-based cell-matrix adhesion in regulating c-Src/FAK signaling under decreased anchorage conditions.     

Activation of c-Src in cells bearing v-Crk and its suppression by Csk.

The protein product of the CT10 virus, p47gag-crk (v-Crk), which contains Src homology region 2 (SH2) and 3 (SH3) domains but lacks a kinase domain, is believed to cause an increase in cellular protein tyrosine phosphorylation. A candidate tyrosine kinase, Csk (C-terminal Src kinase), has been implicated in c-Src Tyr-527 phosphorylation, which negatively regulates the protein tyrosine kinase of pp60c-src (c-Src). To investigate how c-Src kinase activity is regulated in vivo, we first looked at whether v-Crk can activate c-Src kinase. We found that cooverexpression of v-Crk and c-Src caused elevation of c-Src kinase activity, resulting in an increase of tyrosine phosphorylation of cellular proteins and morphological transformation of rat 3Y1 fibroblasts. v-Crk and c-Src complexes were not detected, although v-Crk bound to a variety of tyrosine-phosphorylated proteins in cells overexpressing v-Crk and c-Src. Overexpression of Csk in these transformed cells caused reversion to normal phenotypes and also reduced the level of c-Src kinase activity. However, Csk did not cause reversion of cells transformed by v-Src or c-Src527F, in which Tyr-527 was changed to Phe. These results strongly suggest that Csk acts on Tyr-527 of c-Src and suppresses c-Src kinase activity in vivo. Because Csk can suppress transformation by cooverexpression of v-Crk and c-Src, we suggest that v-Crk causes activation of c-Src in vivo by altering the phosphorylation state of Tyr-527.     

Evidence for in vivo phosphorylation of the Grb2 SH2-domain binding site on focal adhesion kinase by Src-family protein-tyrosine kinases.

Focal adhesion kinase (FAK) is a nonreceptor protein-tyrosine kinase (PTK) that associates with integrin receptors and participates in extracellular matrix-mediated signal transduction events. We showed previously that the c-Src nonreceptor PTK and the Grb2 SH2/SH3 adaptor protein bound directly to FAK after fibronectin stimulation (D. D. Schlaepfer, S.K. Hanks, T. Hunter, and P. van der Geer, Nature [London] 372:786-791, 1994). Here, we present evidence that c-Src association with FAK is required for Grb2 binding to FAK. Using a tryptic phosphopeptide mapping approach, the in vivo phosphorylation of the Grb2 binding site on FAK (Tyr-925) was detected after fibronectin stimulation of NIH 3T3 cells and was constitutively phosphorylated in v-Src-transformed NIH 3T3 cells. In vitro, c-Src phosphorylated FAK Tyr-925 in a glutathione S-transferase-FAK C-terminal domain fusion protein, whereas FAK did not. Using epitope-tagged FAK constructs, transiently expressed in human 293 cells, we determined the effect of site-directed mutations on c-Src and Grb2 binding to FAK. Mutation of FAK Tyr-925 disrupted Grb2 binding, whereas mutation of the c-Src binding site on FAK (Tyr-397) disrupted both c-Src and Grb2 binding to FAK in vivo. These results support a model whereby Src-family PTKs are recruited to FAK and focal adhesions following integrin-induced autophosphorylation and exposure of FAK Tyr-397. Src-family binding and phosphorylation of FAK at Tyr-925 creates a Grb2 SH2-domain binding site and provides a link to the activation of the Ras signal transduction pathway. In Src-transformed cells, this pathway may be constitutively activated as a result of FAK Tyr-925 phosphorylation in the absence of integrin stimulation.     

斑马鱼模型在药物筛选中的应用

斑马鱼具有子代数量多、体外受精、胚胎透明、可以做大规模遗传突变筛选等生物学特性, 因此成为一种良好的脊椎动物模式生物。随着研究的深入, 斑马鱼不仅应用于遗传学和发育生物学研究, 而且拓展和延伸到疾病模型和药物筛选领域。作为一种整体动物模型, 斑马鱼能够全面地检测评估化合物的活性和副作用, 实现高内涵筛选。近年来, 科学家们不断地发展出新的斑马鱼疾病模型和新的筛选技术, 并找到了一批活性化合物。这些化合物大多数在哺乳动物模型中也有相似的效果, 其中前列腺素E2(dmPGE2)和来氟米特(Leflunomide)已经进入临床实验, 分别用来促进脐带血细胞移植后的增殖和治疗黑素瘤。这些成果显示了斑马鱼模型很适合用于药物筛选。文章概括介绍了斑马鱼模型的特点和近年来在疾病模型和药物筛选方面的进展, 希望能够帮助人们了解斑马鱼在新药研发中的应用, 并开展基于斑马鱼模型的药物筛选。     

Small molecule screening in the zebrafish

The zebrafish is an ideal organism for small molecule studies. The ability to use the whole organism allows complex in vivo phenotypes to be assayed and combines animal testing with screening. Embryos are easily treatable by waterborne exposure. The small size and abundance of embryos make zebrafish suitable for screening in a high-throughput manner in 96- or 48-well plates. Zebrafish embryos have successfully been used in chemical genetic screens to elucidate biological pathways and find chemical suppressors. Small molecules discovered by screening zebrafish disease models may also be useful as lead compounds for drug development as there appears to be a high level of conservation of drug activity between mammals and zebrafish. Here we provide the technical aspects of treating embryos with small molecules and performing chemical screens with zebrafish.     

A rapid, high content, in vivo model of glucocorticoid-induced osteoporosis

Glucocorticoid-induced osteoporosis (GIOP) is a major clinical problem given the widespread use of steroids and limited efficacy of biphosphonates. Existing animal models of GIOP are both slow and expensive. Hence, there is a need both for adjunctive modelling systems, as well as more efficacious therapies for the treatment of GIOP. We have addressed this issue through the creation of a zebrafish model of GIOP, which can be used for 96-well plate in vivo screening with an assay time of 5 days. The model demonstrates key similarities to human GIOP including a partial response to bisphosphonates. The ability to extract detailed pharmacological data, including concentration-response analyses, enables the screening and ranking of candidate therapeutic compounds. In addition, the zebrafish model is highly relevant for pathway dissection through genetic knockdown and overexpression studies.     

Homology-modeled ligand-binding domains of zebrafish estrogen receptors alpha, beta1, and beta2: from in silico to in vivo studies of estrogen interactions in Danio rerio as a model system

Homology models were constructed for the ligand-binding domains of zebrafish estrogen receptors (zfERs) alpha, beta(1), and beta(2). Estradiol-binding sites are nearly identical in zfERs and their human homologs, suggesting that zebrafish will serve as a good model system for studying human ER-binding drugs. Conversely, studies of endocrine disruptor effects on zebrafish will benefit from the wealth of data available on xenoestrogen interactions with human ERs. Compounds flagged by the Interagency Coordinating Committee on the Validation of Alternative Methods for endocrine disruptor screening were docked into our zfER homology models. Ideally, these in silico docking studies would be complemented with in vivo binding studies. To this end, fluorescently tagged estradiol was docked into zfERalpha and found to bind in the same manner as in human ERalpha, with fluorescein preferentially occupying a region between helices 11 and 12. Fluorescently tagged estradiol was synthesized and was found to localize along the path of primordial germ cell migration in the developing zebrafish embryo 3 d after fertilization, consistent with previous reports of 1) a role for estradiol in sex determination, and 2) the first appearance of ERs 2 d after fertilization. These data provide a foundation for future in silico and in vivo binding studies of estrogen agonists and antagonists with zebrafish ERs.     

An In Vivo Method to Quantify Lymphangiogenesis in Zebrafish

Background

Lymphangiogenesis is a highly regulated process involved in the pathogenesis of disease. Current in vivo models to assess lymphangiogenesis are largely unphysiologic. The zebrafish is a powerful model system for studying development, due to its rapid growth and transparency during early stages of life. Identification of a network of trunk lymphatic capillaries in zebrafish provides an opportunity to quantify lymphatic growth in vivo.

Methods and Results

Late-phase microangiography was used to detect trunk lymphatic capillaries in zebrafish 2- and 3-days post-fertilization. Using this approach, real-time changes in lymphatic capillary development were measured in response to modulators of lymphangiogenesis. Recombinant human vascular endothelial growth factor (VEGF)-C added directly to the zebrafish aqueous environment as well as human endothelial and mouse melanoma cell transplantation resulted in increased lymphatic capillary growth, while morpholino-based knockdown of vegfc and chemical inhibitors of lymphangiogenesis added to the aqueous environment resulted in decreased lymphatic capillary growth.

Conclusion

Lymphatic capillaries in embryonic and larval zebrafish can be quantified using late-phase microangiography. Human activators and small molecule inhibitors of lymphangiogenesis, as well as transplanted human endothelial and mouse melanoma cells, alter lymphatic capillary development in zebrafish. The ability to rapidly quantify changes in lymphatic growth under physiologic conditions will allow for broad screening of lymphangiogenesis modulators, as well as help define cellular roles and elucidate pathways of lymphatic development.     

Zebrafish: as an integrative model for twenty-first century toxicity testing

The zebrafish embryo is a useful small model for investigating vertebrate development because of its transparency, low cost, transgenic and morpholino capabilities, conservation of cell signaling, and concordance with mammalian developmental phenotypes. From these advantages, the zebrafish embryo has been considered as an alternative model for traditional in vivo developmental toxicity screening. The use of this organism in conjunction with traditional in vivo developmental toxicity testing has the potential to reduce cost and increase throughput of testing the chemical universe, prioritize chemicals for targeted toxicity testing, generate predictive models of developmental toxicants, and elucidate mechanisms and adverse outcome pathways for abnormal development. This review gives an overview of the zebrafish embryo for pre dictive toxicology and 21st century toxicity testing. Developmental eye defects were selected as an example to evaluate data from the U.S. Environmental Protection Agency's ToxCast program comparing responses in zebrafish embryos with those from pregnant rats and rabbits for a subset of 24 environmental chemicals across >600 in vitro assay targets. Cross-species comparisons implied a common basis for biological pathways associated with neuronal defects, extracellular matrix remodeling, and mitotic arrest.     

The transmembrane adaptor Cbp/PAG1 controls the malignant potential of human non-small cell lung cancers that have c-src upregulation

The tyrosine kinase c-Src is upregulated in various human cancers, although the precise regulatory mechanism underlying this upregulation is unclear. We previously reported that a transmembrane adaptor Csk-binding protein (Cbp; PAG1) plays an important role in controlling the cell transformation that is induced by the activation of c-Src. To elucidate the in vivo role of Cbp, we examined the function of Cbp in lung cancer cell lines and tissues. In this study, we found that Cbp was markedly downregulated in human non-small cell lung cancer (NSCLC) cells. The ectopic expression of Cbp suppressed the anchorage-independent growth of the NSCLC cell lines (A549 and Lu99) that had upregulated c-Src, whereas the Cbp expression had little effect on other NSCLC cell lines (PC9 and Lu65) that express normal levels of c-Src. The expression of Cbp suppressed the kinase activity of c-Src in A549 cells by recruiting c-Src and its negative regulator, C-terminal Src kinase (Csk), to lipid rafts. The treatment with Src inhibitors, such as PP2, dasatinib, and saracatinib, also suppressed the growth of A549 cells. Furthermore, Cbp expression attenuated the ability of A549 cells to form tumors in nude mice, invade in vitro, and metastasize in vivo. In addition, we found a significant inverse correlation between the level of Cbp expression and the extent of lymph node metastasis in human lung cancers. These results indicate that Cbp is required for the Csk-mediated inactivation of c-Src and may control the promotion of malignancy in NSCLC tumors that are characterized by c-Src upregulation.     

A quantitative peptide array for evaluation of protein kinase activity

Peptide array, which is known as an emerging technology, has been developed for identification of protein kinase activity. For this purpose, the ability of quantitative analysis is very important because the absolute change in protein kinase activity is critical for the determination of cellular function. Here we report an original type of peptide array for quantitative evaluation of protein kinase activity by fluorescence imaging. We used the peptide array for the quantitative evaluation of the nonreceptor tyrosine kinase c-Src activity as a model for detecting protein kinase activities. By using positive and negative control peptides, we obtained the actual ratio of tyrosine phosphorylation of substrate peptide not only by purified c-Src but also by c-Src in cell lysate. In addition, the experimental approach provided simple immobilization of peptide. Our sensitive, specific, and high-throughput peptide array can be used for quantitative evaluation of kinase activity and potentially can be applied to drug discovery and screening.     

Studying synthetic lethal interactions in the zebrafish system: insight into disease genes and mechanisms

The post-genomic era is marked by a pressing need to functionally characterize genes through understanding gene-gene interactions, as well as interactions between biological pathways. Exploiting a phenomenon known as synthetic lethality, in which simultaneous loss of two interacting genes leads to loss of viability, aids in the investigation of these interactions. Although synthetic lethal screening is a powerful technique that has been used with great success in many model organisms, including Saccharomyces cerevisiae, Drosophila melanogaster and Caenorhabditis elegans, this approach has not yet been applied in the zebrafish, Danio rerio. Recently, the zebrafish has emerged as a valuable system to model many human disease conditions; thus, the ability to conduct synthetic lethal screening using zebrafish should help to uncover many unknown disease-gene interactions. In this article, we discuss the concept of synthetic lethality and provide examples of its use in other model systems. We further discuss experimental approaches by which the concept of synthetic lethality can be applied to the zebrafish to understand the functions of specific genes.