Gabor Kari

Zebrafish : An emerging model system for human disease and drug discovery

In vivo studies represent an essential step in drug development and currently rely largely on mice, yet limitations of mammalian models motivated the search for complementary vertebrate model systems. This review focuses on zebrafish, Danio rerio, as a facile model system to study human disease and drug responses. Zebrafish are particularly suited for this purpose because they represent a vertebrate species, their genome is sequenced, and a large number of synchronously developing, transparent embryos can be produced. Zebrafish embryos are permeable to drugs and can easily be manipulated using well‐established genetic and molecular approaches. Here, we summarize recent work on drug discovery and toxicity in zebrafish embryos. In addition, we provide a synopsis of current efforts to establish disease models in zebrafish focusing on neoplasia. The results of these studies highlight the potential of zebrafish as a viable addition to established animal models by offering medium and, potentially, high throughput capabilities.

In vivo Radioprotection by the Fullerene Nanoparticle DF-1 as Assessed in a Zebrafish Model

Purpose: We have previously shown that zebrafish (Danio rerio) embryos can be used as an in vivo model to validate modifiers of the radiation response. Here, we evaluated the radioprotective effect of the nanoparticle DF-1, a fullerene with antioxidant properties, in zebrafish embryos. Experimental Design: Zebrafish embryos were exposed to different doses of ionizing radiation ranging from 20 to 80 Gy in the presence and absence of DF-1. Toxicity and radioprotective effects were assessed by monitoring overall survival and morphology as well as organ functions by employing assays to measure kidney excretory function and development of sensory nerve cells (neuromasts). Antioxidant properties of DF-1 were assessed in whole fish. Results: DF-1 had no apparent adverse effects on normal zebrafish morphology or viability throughout the concentration range tested (1-1,000 μmol/L). Ionizing radiation (10-40 Gy) caused time-dependent and dose-dependent perturbations of normal zebrafish morphology and physiology, notably defective midline development resulting in dorsal curvature of the body axis (“curly-up”), neurotoxicity, impaired excretory function, and decreased survival of the exposed embryos. DF-1 (100 μmol/L) markedly attenuated overall and organ-specific radiation-induced toxicity when given within 3 hours before or up to 15 minutes after radiation exposure. By contrast, DF-1 afforded no protection when given 30 minutes after ionizing radiation. The degree of radioprotection provided by DF-1 was comparable with that provided by the Food and Drug Administration–approved radioprotector amifostine (4 mmol/L). Protection against radiation-associated toxicity using DF-1 in zebrafish embryos was associated with marked reduction of radiation-induced reactive oxygen species. Conclusion: The fullerene DF-1 protects zebrafish embryos against deleterious effects of ionizing radiation due, in part, to its antioxidant properties.

A Novel Preclinical Strategy for Identifying Cardiotoxic Kinase Inhibitors and Mechanisms of Cardiotoxicity

Rationale: Despite intense interest in strategies to predict which kinase inhibitor (KI) cancer therapeutics may be associated with cardiotoxicity, current approaches are inadequate. Sorafenib is a KI of concern because it inhibits growth factor receptors and Raf-1/B-Raf, kinases that are upstream of extracellular signal-regulated kinases (ERKs) and signal cardiomyocyte survival in the setting of stress. Objectives: To explore the potential use of zebrafish as a preclinical model to predict cardiotoxicity and to determine whether sorafenib has associated cardiotoxicity, and, if so, define the mechanisms. Methods and Results: We find that the zebrafish model is readily able to discriminate a KI with little or no cardiotoxicity (gefitinib) from one with demonstrated cardiotoxicity (sunitinib). Sorafenib, like sunitinib, leads to cardiomyocyte apoptosis, a reduction in total myocyte number per heart, contractile dysfunction, and ventricular dilatation in zebrafish. In cultured rat cardiomyocytes, sorafenib induces cell death. This can be rescued by adenovirus-mediated gene transfer of constitutively active MEK1, which restores ERK activity even in the presence of sorafenib. Whereas growth factor–induced activation of ERKs requires Raf, &agr;-adrenergic agonist-induced activation of ERKs does not require it. Consequently, activation of &agr;-adrenergic signaling markedly decreases sorafenib-induced cell death. Consistent with these in vitro data, inhibition of &agr;-adrenergic signaling with the receptor antagonist prazosin worsens sorafenib-induced cardiomyopathy in zebrafish. Conclusions: Zebrafish may be a valuable preclinical tool to predict cardiotoxicity. The &agr;-adrenergic signaling pathway is an important modulator of sorafenib cardiotoxicity in vitro and in vivo and appears to act through a here-to-fore unrecognized signaling pathway downstream of &agr;-adrenergic activation that bypasses Raf to activate ERKs.

Interaction with LC8 is required for Pak1 nuclear import and is indispensable for zebrafish development.

Pak1 (p21 activated kinase 1) is a serine/threonine kinase implicated in regulation of cell motility and survival and in malignant transformation of mammary epithelial cells. In addition, the dynein light chain, LC8, has been described to cooperate with Pak1 in malignant transformation of breast cancer cells. Pak1 itself may aid breast cancer development by phosphorylating nuclear proteins, including estrogen receptor alpha. Recently, we showed that the LC8 binding site on Pak1 is adjacent to the nuclear localization sequence (NLS) required for Pak1 nuclear import. Here, we demonstrate that the LC8-Pak1 interaction is necessary for epidermal growth factor (EGF)-induced nuclear import of Pak1 in MCF-7 cells, and that this event is contingent upon LC8-mediated Pak1 dimerization. In contrast, Pak2, which lacks an LC8 binding site but contains a nuclear localization sequence identical to that in Pak1, remains cytoplasmic upon EGF stimulation of MCF-7 cells. Furthermore, we show that severe developmental defects in zebrafish embryos caused by morpholino injections targeting Pak are partially rescued by co-injection of wild-type human Pak1, but not by co-injection of mutant Pak1 mRNA disrupting either the LC8 binding or the NLS site. Collectively, these results suggest that LC8 facilitates nuclear import of Pak1 and that this function is indispensable during vertebrate development.

Inhibition of p73 function by Pifithrin-α as revealed by studies in zebrafish embryos

The p53 family of proteins contains two members that have been implicated in sensitization of cells and organisms to genotoxic stress, i.e., p53 itself and p73. In vitro, lack of either p53 or p73 can protect certain cell types in the adult organism against death upon exposure to DNA damaging agents. The present study was designed to assess the relative contribution of p53 to radiation resistance of an emerging vertebrate model organism, i.e., zebrafish embryos. Consistent with previous reports, suppressing p53 protein expression using antisense morpholino oligonucleotides (MOs) increased survival and reduced gross morphological alterations in zebrafish embryos exposed to ionizing radiation. By contrast, a pharmacological inhibitor of p53 function (Pifithrin-α (PFT-α)) caused developmental abnormalities affecting the head, brain, eyes and kidney function and did not protect against lethal effects of ionizing radiation when administered at 3 hours post fertilization (hpf). The phenotypic abnormalities associated with PFT-α treatment were similar to those caused by antisense MO knock down (kd) used to reduce p73 expression. PFT-α also inhibited p73-dependent transcription of a reporter gene construct containing canonical p53-responsive promoter sequences. Notably, when administered at later stages of development (23 hpf), PFT-α did not cause overt developmental defects but exerted radioprotective effects in zebrafish embryos. In summary, this study highlights off-target effects of the pharmacological p53 inhibitor PFT-α related to inhibition of p73 function and essential roles of p73 at early but not later stages of zebrafish development.

Nuclear factor kappaB inhibitors alleviate and the proteasome inhibitor PS-341 exacerbates radiation toxicity in zebrafish embryos.

Inflammatory changes are a major component of the normal tissue response to ionizing radiation, and increased nuclear factor κB (NF-κB) activity is an important mediator of inflammatory responses. Here, we used zebrafish embryos to assess the capacity of two different classes of pharmacologic agents known to target NF-κB to modify radiation toxicity in the vertebrate organism. These were proteasome inhibitors, including lactacystin, MG132, and PS-341 (Bortezomib/VELCADE), and direct inhibitors of NF-κB activity, including ethyl pyruvate (EP) and the synthetic triterpenoid CDDO-TFEA (RTA401), among others. The proteasome inhibitors either did not significantly affect radiation sensitivity of zebrafish embryos (MG132, lactacystin) or rendered zebrafish embryos more sensitive to the lethal effects of ionizing radiation (PS-341). Radiosensitization by PS-341 was reduced in fish with impaired p53 expression or function but not associated with enhanced expression of select p53 target genes. In contrast, the direct NF-κB inhibitors EP and CDDO-TFEA significantly improved overall survival of lethally irradiated zebrafish embryos. In addition, direct NF-κB inhibition reduced radiation-induced apoptosis in the central nervous system, abrogated aberrations in body axis development, restored metabolization and secretion of a reporter lipid through the gastrointestinal system, and improved renal clearance compromised by radiation. In contrast to amifostine, EP and CDDO-TFEA not only protected against but also mitigated radiation toxicity when given 1 to 2 hours postexposure. Finally, four additional IκB kinase inhibitors with distinct mechanisms of action similarly improved overall survival of lethally irradiated zebrafish embryos. In conclusion, inhibitors of canonical pathways to NF-κB activation may be useful in alleviating radiation toxicity in patients. [Mol Cancer Ther 2009;8(9):2625–34]

Nuclear Factor κB Inhibitors Alleviate and the Proteasome Inhibitor PS-341 Exacerbates Radiation Toxicity in Zebrafish Embryos

Inflammatory changes are a major component of the normal tissue response to ionizing radiation, and increased nuclear factor κB (NF-κB) activity is an important mediator of inflammatory responses. Here, we used zebrafish embryos to assess the capacity of two different classes of pharmacologic agents known to target NF-κB to modify radiation toxicity in the vertebrate organism. These were proteasome inhibitors, including lactacystin, MG132, and PS-341 (Bortezomib/VELCADE), and direct inhibitors of NF-κB activity, including ethyl pyruvate (EP) and the synthetic triterpenoid CDDO-TFEA (RTA401), among others. The proteasome inhibitors either did not significantly affect radiation sensitivity of zebrafish embryos (MG132, lactacystin) or rendered zebrafish embryos more sensitive to the lethal effects of ionizing radiation (PS-341). Radiosensitization by PS-341 was reduced in fish with impaired p53 expression or function but not associated with enhanced expression of select p53 target genes. In contrast, the direct NF-κB inhibitors EP and CDDO-TFEA significantly improved overall survival of lethally irradiated zebrafish embryos. In addition, direct NF-κB inhibition reduced radiation-induced apoptosis in the central nervous system, abrogated aberrations in body axis development, restored metabolization and secretion of a reporter lipid through the gastrointestinal system, and improved renal clearance compromised by radiation. In contrast to amifostine, EP and CDDO-TFEA not only protected against but also mitigated radiation toxicity when given 1 to 2 hours postexposure. Finally, four additional IκB kinase inhibitors with distinct mechanisms of action similarly improved overall survival of lethally irradiated zebrafish embryos. In conclusion, inhibitors of canonical pathways to NF-κB activation may be useful in alleviating radiation toxicity in patients. [Mol Cancer Ther 2009;8(9):2625-34] Introduction Normal tissue damage limits the dose of ionizing radiation that can be safely administered to treat neoplastic disease. A well-known example of this problem is inflammation of the oral mucosa and of the lining of the gastrointestinal tract in tumor patients receiving chemotherapy or radiation.1 Depending on the area of the body treated with radiation, other organ sites including the lungs and the pericardium also manifest radiation-induced inflammation. A pervasive feature of ionizing radiation–associated inflammation is the increased presence of proinflammatory cytokines including tumor necrosis factor-α (TNF-α) and interleukin-6, both locally and in the circulation.2 In contrast to intracellular regulators of the DNA damage response, these and other inflammatory mediators act in a paracrine fashion affecting diverse cell types in the tissue microenvironment or even at a distance.3 This circumstance highlights the necessity to use animal models to investigate the relative contribution of inflammatory changes to the overall response to radiation-induced cell and tissue injury in a multicellular organism. In recognition of this need, we recently established zebrafish embryos as a facile vertebrate in vivo system to monitor the effects of radiation protectors on normal tissues during development.4 The nuclear factor κB (NF-κB) family of transcription factors represents a diverse and shared signaling mechanism activated during cell stress responses.5 In addition, deregulated NF-κB signaling has been implicated in the malignant phenotype and treatment resistance of select tumor forms.6-10 The canonical pathway to NF-κB activation leads to IκB kinase β (IKKβ)-dependent phosphorylation and subsequent proteasomal degradation of the NF-κB inhibitor IκB, increased nuclear presence of NF-κB dimers, and enhanced NF-κB– dependent transcriptional activity.5 Whole-body radioprotection through antiinflammatory agents has very recently been shown in animal models. Specifically, certain triterpenoids (CDDO and derivatives thereof) have been shown to selectively protect normal mouse tissues against the deleterious effects of ionizing radiation.11 Furthermore, ethyl pyruvate (EP), a derivative of the end product of glycolysis, similarly protects normal cells against the deleterious effects of radiation both in vitro and in mice.12 Among other molecular targets, both drugs inhibit activation of NF-κB. EP inhibits NF-κB signaling through direct molecular interaction with a reactive cysteine of the p65 subunit of NF-κB13 whereas CDDOTFEA binds to a reactive cysteine (Cys179) of IKKα, thus inhibiting its kinase activity.14 However, these drugs also target other signaling molecules and pathways of potential relevance to the radiation response, including signal transducers and activators of transcription 3 and Jaks.15, 16 In addition to these agents proteasome inhibitors have been shown to inhibit NF-κB–dependent transcription, and one of these (PS-341; Bortezomib; VELCADE) Studies Borbala Daroczi,1 Gabor Kari,1 Qing Ren,1 Adam P. Dicker,1,3 and Ulrich Rodeck2,3 1Departments of Radiation Oncology, and 2Dermatology and Cutaneous Biology, and 3Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania Reprinted with permission from the American Association of Cancer Research, “Nuclear factor κB inhibitors alleviate and the proteasome inhibitor PS-341 exacerbates radiation toxicity in zebrafish embryos”, Moleculaer Cancer Therapy, 2009;8(9), pages 2625-2634.