RESUMO
There is a need for novel therapies targeting hypoxic cells in tumors. These cells are associated with tumor resistance to therapy and express hypoxia inducible factor-1 (HIF-1), a transcription factor that mediates metabolic adaptation to hypoxia and activates tumor angiogenesis. We previously developed an oncolytic adenovirus (HYPR-Ad) for the specific killing of hypoxic/HIF-active tumor cells, which we now armed with an interleukin-4 gene (HYPR-Ad-IL4). We designed HYPR-Ad-IL4 by cloning the Ad E1A viral replication and IL-4 genes under the regulation of a bidirectional hypoxia/HIF-responsive promoter. The IL-4 cytokine was chosen for its ability to induce a strong host antitumor immune response and its potential antiangiogenic activity. HYPR-Ad-IL4 induced hypoxia-dependent IL-4 expression, viral replication, and conditional cytolysis of hypoxic, but not normoxic cells. The treatment of established human tumor xenografts with HYPR-Ad-IL4 resulted in rapid and maintained tumor regression with the same potency as that of wild-type dl309-Ad. HYPR-Ad-IL4-treated tumors displayed extensive necrosis, fibrosis, and widespread viral replication. Additionally, these tumors contained a distinctive leukocyte infiltrate and prominent hypoxia. The use of an oncolytic Ad that locally delivers IL-4 to tumors is novel, and we expect that HYPR-Ad-IL4 will have broad therapeutic use for all solid tumors that have hypoxia or active HIF, regardless of tissue origin or genetic alterations.
Assuntos
Adenoviridae/metabolismo , Terapia Genética/métodos , Fator 1 Induzível por Hipóxia/metabolismo , Interleucina-4/genética , Interleucina-4/metabolismo , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Clonagem Molecular , Humanos , Hipóxia , Camundongos , Modelos Genéticos , Transplante de NeoplasiasRESUMO
The commercially available Jak2 inhibitor, alpha-cyano-3,4-dihydroxy-N-benzylcinnamide (AG490), has been used extensively to study Jak2 kinase function. While alpha-cyano-3,4-dihydroxy-N-benzylcinnamide is a potent Jak2 inhibitor, it can inhibit a number of other kinase signaling pathways as well. To circumvent this problem, we sought to identify novel small molecule inhibitors of Jak2 tyrosine kinase activity. For this, we constructed a homology model of the Jak2 kinase domain and identified solvent accessible pockets on the surface of the structure. Using the DOCK program, we tested 6451 compounds of known chemical structure in silico for their ability to interact with a pocket positioned adjacent to the activation loop. We attained the top seven scoring compounds from the National Cancer Institute and tested their ability to inhibit Jak2 autophosphorylation in vitro. Using Western blot analysis, we found that one of the compounds, 1,2,3,4,5,6-hexabromocyclohexane, was able to potently, and directly, inhibit Jak2 autophosphorylation. Characterization of this compound revealed that it inhibits Jak2 tyrosine autophosphorylation in both a time- and concentration-dependent manner. It greatly reduced growth hormone-mediated Jak2 autophosphorylation but did not block autophosphorylation of the epidermal growth factor receptor. Furthermore, doses as high as 100 muM were not toxic to cells as measured by their ability to exclude propidium iodide. As such, we believe that this compound could serve as a lead compound for a new generation of Jak2 inhibitors and, perhaps, be useful in elucidating the mechanisms of Jak2 kinase function.
Assuntos
Cicloexanos/química , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/química , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/química , Animais , Sítios de Ligação , Linhagem Celular , Cicloexanos/farmacologia , Bases de Dados Factuais , Haplorrinos , Janus Quinase 2 , Ligantes , Modelos Moleculares , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Solventes , Relação Estrutura-AtividadeRESUMO
Traditionally, cell adhesion assays are performed in a manual workstation format using fluorescence-based readouts. Herein, the authors describe a label-free homogeneous assay to identify inhibitors of α4ß7 integrin-mediated cell adhesion to its ligand, the mucosal addressin cell adhesion molecule (MadCAM), using the SRU BIND platform. The biosensor is optically based and comprises a subwavelength polymer grating. The assay was validated using standard compounds and an α4 blocking antibody and correlated very closely with the manual assay format when running a battery of test compounds of varying potencies. Cell adhesion was strictly dependent on the presence of divalent cations where Mg(2+) was greater than Ca(2+) at promoting cell adhesion. This homogeneous and label-free format exhibited low variability with a calculated Z' of 0.6. In addition to measuring α4ß7-mediated 8866 cell adhesion to MadCAM, the authors also demonstrate that this platform can measure adhesion of Jurkat cells expressing α4ß1 to the vascular cell adhesion molecule. Thus, the SRU BIND platform is widely applicable to measuring cell adhesion events mediated by other integrins binding to their receptors in an assay format that is amenable to high-throughput screening.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Imunoglobulinas/metabolismo , Integrinas/antagonistas & inibidores , Integrinas/metabolismo , Mucoproteínas/metabolismo , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular , Linhagem Celular , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Células Jurkat , Cinética , Metais/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Angiogenesis is a critical physiologic process that is appropriated during tumorigenesis. Little is known about how this process is specifically regulated in the brain. Brain angiogenesis inhibitor-1 (BAI1) is a brain-predominant seven-transmembrane protein that contains five antiangiogenic thrombospondin type-1 repeats (TSR). We recently showed that BAI1 is cleaved at a conserved proteolytic cleavage site releasing a soluble, 120 kDa antiangiogenic factor called vasculostatin (Vstat120). Vstat120 has been shown to inhibit in vitro angiogenesis and suppress subcutaneous tumor growth. Here, we examine its effect on the intracranial growth of malignant gliomas and further study its antitumor mechanism. First, we show that expression of Vstat120 strongly suppresses the intracranial growth of malignant gliomas, even in the presence of the strong proangiogenic stimulus mediated by the oncoprotein epidermal growth factor receptor variant III (EGFRvIII). This tumor-suppressive effect is accompanied by a decrease in tumor vascular density, suggesting a potent antiangiogenic effect in the brain. Second, and consistent with this interpretation, we find that treatment with Vstat120 reduces the migration of cultured microvascular endothelial cells in vitro and inhibits corneal angiogenesis in vivo. Third, we show that these antivascular effects critically depend on the presence of the cell surface receptor CD36 on endothelial cells in vitro and in vivo, supporting the role of Vstat120 TSRs in mediating these effects. These results advance the understanding of brain-specific angiogenic regulation, and suggest that Vstat120 has therapeutic potential in the treatment of brain tumors and other intracerebral vasculopathies.
Assuntos
Proteínas Angiogênicas/biossíntese , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/terapia , Antígenos CD36/metabolismo , Glioblastoma/irrigação sanguínea , Glioblastoma/terapia , Fragmentos de Peptídeos/biossíntese , Proteínas Angiogênicas/genética , Proteínas Angiogênicas/metabolismo , Animais , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Neovascularização da Córnea/tratamento farmacológico , DNA Complementar/administração & dosagem , DNA Complementar/genética , Células Endoteliais/patologia , Glioblastoma/genética , Humanos , Camundongos , Camundongos Nus , Neovascularização Patológica/metabolismo , Neovascularização Patológica/terapia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ratos , Receptores Acoplados a Proteínas G , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Pathologic angiogenesis has emerged as an important therapeutic target in several major diseases. Zebrafish offer the potential for high-throughput drug discovery in a whole vertebrate system. We developed the first quantitative, automated assay for antiangiogenic compound identification using zebrafish embryos. This assay uses transgenic zebrafish with fluorescent blood vessels to facilitate image analysis. We developed methods for automated drugging and imaging of zebrafish in 384-well plates and developed a custom algorithm to quantify the number of angiogenic blood vessels in zebrafish. The assay was used to screen the LOPAC1280 compound library for antiangiogenic compounds. Two known antiangiogenic compounds, SU4312 and AG1478, were identified as hits. Additionally, one compound with no previously known antiangiogenic activity, indirubin-3'-monoxime (IRO), was identified. We showed that each of the hit compounds had dose-dependent antiangiogenic activity in zebrafish. The IC(50) of SU4312, AG1478, and IRO in the zebrafish angiogenesis assay was 1.8, 8.5, and 0.31 micromol/L, respectively. IRO had the highest potency of the hit compounds. Moreover, IRO inhibited human umbilical vein endothelial cell tube formation and proliferation (IC(50) of 6.5 and 0.36 micromol/L, respectively). It is therefore the first antiangiogenic compound discovered initially in a zebrafish assay that also has demonstrable activity in human endothelial cell-based angiogenesis assays.
Assuntos
Inibidores da Angiogênese/farmacologia , Automação , Avaliação Pré-Clínica de Medicamentos , Endotélio Vascular/efeitos dos fármacos , Indóis/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Oximas/farmacologia , Peixe-Zebra/imunologia , Algoritmos , Animais , Animais Geneticamente Modificados , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/imunologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Embrião não Mamífero/citologia , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/imunologia , Humanos , Indóis/química , Timidina , Veias Umbilicais/citologia , Veias Umbilicais/efeitos dos fármacos , Veias Umbilicais/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismoRESUMO
In vascular smooth muscle cells, Jak2 tyrosine kinase becomes activated in response to oxidative stress in the form of hydrogen peroxide. Although it has been postulated that hydrogen peroxide-induced Jak2 activation promotes cell survival, this has never been tested. We therefore examined the role that Jak2 plays in vascular smooth muscle cell apoptosis following hydrogen peroxide treatment. Here, we report that Jak2 tyrosine kinase activation by hydrogen peroxide is required for apoptosis of vascular smooth muscle cells. Upon treatment of primary rat aortic smooth muscle cells with hydrogen peroxide, we observed laddering of genomic DNA and nuclear condensation, both hallmarks of apoptotic cells. However, apoptosis was prevented by either the expression of a dominant negative Jak2 protein or by the Jak2 pharmacological inhibitor AG490. Moreover, expression of the proapoptotic Bax protein was induced following hydrogen peroxide treatment. Again, expression of a dominant negative Jak2 protein or treatment of cells with AG490 prevented this Bax induction. Following Bax induction by hydrogen peroxide, mitochondrial membrane integrity was compromised, and caspase-9 became activated. In contrast, in cells expressing a Jak2 dominant negative we observed that mitochondrial membrane integrity was preserved, and no caspase-9 activation occurred. These data demonstrate that the activation of Jak2 tyrosine kinase by hydrogen peroxide is essential for apoptosis of vascular smooth muscle cells. Furthermore, this report identifies Jak2 as a potential therapeutic target in vascular diseases in which vascular smooth muscle cell apoptosis contributes to pathological progression.
Assuntos
Apoptose , Músculo Liso Vascular/citologia , Estresse Oxidativo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Animais , Western Blotting , Caspase 9 , Caspases/metabolismo , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Sobrevivência Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Ativação Enzimática , Genes Dominantes , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/farmacologia , Janus Quinase 2 , Mitocôndrias/metabolismo , Testes de Precipitina , Proteínas Proto-Oncogênicas/metabolismo , Ratos , Fatores de Tempo , Tirfostinas/farmacologia , Regulação para Cima , Proteína X Associada a bcl-2RESUMO
Angiotensin II is a well-known vasoactive peptide, but it can also act as a potent growth factor, partially through activation of the tyrosine kinase Jak2. Activated Jak2 tyrosine phosphorylates and activates members of the Signal Transducers and Activators of Transcription (STAT) family of cytoplasmic transcription factors. Recently, we demonstrated that tryptophan 1020 and glutamic acid 1024 within the Jak2 activation loop are required for Jak2 tyrosine kinase activity. Here, we sought to elucidate the requirement of glutamic acid 1024 for Jak2 function. Using molecular modeling algorithms of the Jak2 kinase domain, we identified a putative interaction between glutamic acid 1024 and an arginine at position 1113. We generated a series of charge-based substitution mutations at position 1113 and found that conversion of arginine 1113 to glutamic acid, alanine, or lysine prevented Jak2 autophosphorylation. Furthermore, mutation of arginine 1113 prevented the following angiotensin II-dependent processes from occurring: (1) Jak2 tyrosine phosphorylation, (2) Jak2/AT1receptor co-association, (3) STAT1 recruitment to the Jak2/AT1receptor complex, (4) STAT1 tyrosine phosphorylation, and (5) STAT-mediated gene expression. We determined that the interaction between glutamic acid 1024 and arginine 1113 consists of two distinct hydrogen bonds. We conclude that these hydrogen bond interactions are critical for Jak2 kinase function and subsequent angiotensin II-dependent activation of the Jak/STAT signaling pathway.
Assuntos
Arginina/química , Proteínas de Ligação a DNA/metabolismo , Ácido Glutâmico/química , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/fisiologia , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/fisiologia , Transativadores/metabolismo , Alanina/química , Algoritmos , Sequência de Aminoácidos , Angiotensina II/química , Animais , Western Blotting , Células COS , Linhagem Celular , Cristalografia por Raios X , Citoplasma/metabolismo , Regulação da Expressão Gênica , Genes Dominantes , Ligação de Hidrogênio , Imunoprecipitação , Janus Quinase 2 , Luciferases/metabolismo , Lisina/química , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Fosforilação , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fator de Transcrição STAT1 , Transdução de Sinais , Relação Estrutura-Atividade , Transfecção , Tirosina/químicaRESUMO
Previous work has shown that inhibition of Jak2 via the pharmacological compound AG490 blocks the angiotensin II (Ang II)-dependent activation of ERK2, thereby suggesting an essential role of Jak2 in ERK activation. However, recent studies have thrown into question the specificity of AG490 and therefore the role of Jak2 in ERK activation. To address this, we reconstituted an Ang II signaling system in a Jak2-/-cell line and measured the ability of Ang II to activate ERK2 in these cells. Controls for this study were the same cells expressing Jak2 via the addition of a Jak2 expression plasmid. In the cells expressing Jak2, Ang II induced a marked increase in ERK2 activity as measured by Western blot analysis and in vitro kinase assays. ERK2 activity returned to basal levels within 30 min. However, in the cells lacking Jak2, Ang II treatment resulted in ERK2 activation that did not return to basal levels until 120 min after ligand addition. Analysis of phosphatase gene expression revealed that Ang II induced mitogen-activated protein kinase phosphatase 1 (MKP-1) expression in cells expressing Jak2 but failed to induce MKP-1 expression in cells lacking Jak2. Therefore, our results suggest that Jak2 is not required for Ang II-induced ERK2 activation. Rather Jak2 is required for Ang II-induced ERK2 inactivation via induction of MKP-1 gene expression.
Assuntos
Angiotensina II/farmacologia , Proteínas de Ciclo Celular , Proteínas Imediatamente Precoces/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Fosfoproteínas Fosfatases , Proteínas Tirosina Fosfatases/fisiologia , Proteínas Tirosina Quinases/fisiologia , Proteínas Proto-Oncogênicas , Linhagem Celular , Núcleo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fosfatase 1 de Especificidade Dupla , Ativação Enzimática , Humanos , Proteínas Imediatamente Precoces/genética , Janus Quinase 2 , Paxilina , Fosfoproteínas/metabolismo , Fosforilação , Proteína Fosfatase 1 , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Proteínas Tirosina Fosfatases/genética , Receptor Tipo 1 de Angiotensina/fisiologia , Fator de Transcrição STAT1 , Transativadores/metabolismo , Transcrição GênicaRESUMO
Discovered roughly 10 yr ago, Jak2 tyrosine kinase has emerged as a critical molecule in mammalian development, physiology, and disease. Here, we review the early history of Jak2 and its role in health and disease. We will also review its critical role in mediating cytokine-dependent signal transduction. Additionally, more recent work demonstrating the importance of Jak2 in G protein-coupled receptor and tyrosine kinase growth factor receptor signal transduction will be discussed. The cellular and biochemical mechanisms by which Jak2 tyrosine kinase is activated and regulated within the cell also will be reviewed. Finally, structure-function and pharmacological-based studies that identified structural motifs and amino acids within Jak2 that are critical for its function will be examined. By reviewing the biology of Jak2 tyrosine kinase at the molecular, cellular, and physiological levels, we hope to advance the understanding of how a single gene can have such a profound impact on development, physiology, and disease.
Assuntos
Janus Quinase 2/fisiologia , Transcrição Gênica , Motivos de Aminoácidos , Animais , Núcleo Celular/metabolismo , Citocinas/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Janus Quinase 2/metabolismo , Ligantes , Camundongos , Modelos Biológicos , Fosforilação , Estrutura Terciária de Proteína , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Jak2 is a member of the Janus family of tyrosine kinases and is known to be activated by a wide variety of ligands. Here, we sought to identify amino acid residues within Jak2 that are essential for its activation. We provide evidence that glutamic acid 1046 (E1046) is one such residue. Using molecular modeling algorithms of the Jak2 kinase domain, we identified a putative molecular interaction between E1046 and tryptophan 1020 (W1020). Conversion of E1046 to either arginine (E 1046R), alanine (E1046A), aspartic acid (E1046D) or glutamine (E1046Q) abolished Jak2 kinase activity as measured by autophosphorylation assays. Conversion of W1020 to glycine (W1020G) similarly abolished Jak2 kinase activity. Finally, we tested the ability of the E1046R mutant to activate the Jak/STAT signaling pathway in a ligand-dependent signaling system. The ability of angiotensin II to activate the Jak/STAT signaling pathway in cells expressing the E1046R mutant was severely compromised as measured by reduced (1) Jak2 autophosphorylation (2) Jak2 kinase activity (3) AT1/Jak2 co-association (4) Stat1 tyrosine phosphorylation and (5) angiotensin Il-mediated gene transcription. Thus, these studies demonstrate for the first time, the critical role of E1046 in mediating Jak2 activation and its subsequent downstream signaling events.