Adaptation to TKI Treatment Reactivates ERK Signaling in Tyrosine Kinase-Driven Leukemias and Other Malignancies.

Science.gov (United States)

Bruner, J Kyle; Ma, Hayley S; Li, Li; Qin, Alice Can Ran; Rudek, Michelle A; Jones, Richard J; Levis, Mark J; Pratz, Keith W; Pratilas, Christine A; Small, Donald

2017-10-15

FMS-like tyrosine kinase-3 (FLT3) tyrosine kinase inhibitors (TKI) have been tested extensively to limited benefit in acute myeloid leukemia (AML). We hypothesized that FLT3/internal tandem duplication (ITD) leukemia cells exhibit mechanisms of intrinsic signaling adaptation to TKI treatment that are associated with an incomplete response. Here, we identified reactivation of ERK signaling within hours following treatment of FLT3/ITD AML cells with selective inhibitors of FLT3. When these cells were treated with inhibitors of both FLT3 and MEK in combination, ERK reactivation was abrogated and anti-leukemia effects were more pronounced compared with either drug alone. ERK reactivation was also observed following inhibition of other tyrosine kinase-driven cancer cells, including EGFR-mutant lung cancer, HER2-amplified breast cancer, and BCR-ABL leukemia. These studies reveal an adaptive feedback mechanism in tyrosine kinase-driven cancers associated with reactivation of ERK signaling in response to targeted inhibition. Cancer Res; 77(20); 5554-63. ©2017 AACR . ©2017 American Association for Cancer Research.

Role of Receptor Tyrosine Kinase Signaling in Renal Fibrosis

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Feng Liu

2016-06-01

Full Text Available Renal fibrosis can be induced in different renal diseases, but ultimately progresses to end stage renal disease. Although the pathophysiologic process of renal fibrosis have not been fully elucidated, it is characterized by glomerulosclerosis and/or tubular interstitial fibrosis, and is believed to be caused by the proliferation of renal inherent cells, including glomerular epithelial cells, mesangial cells, and endothelial cells, along with defective kidney repair, renal interstitial fibroblasts activation, and extracellular matrix deposition. Receptor tyrosine kinases (RTKs regulate a variety of cell physiological processes, including metabolism, growth, differentiation, and survival. Many studies from in vitro and animal models have provided evidence that RTKs play important roles in the pathogenic process of renal fibrosis. It is also showed that tyrosine kinases inhibitors (TKIs have anti-fibrotic effects in basic research and clinical trials. In this review, we summarize the evidence for involvement of specific RTKs in renal fibrosis process and the employment of TKIs as a therapeutic approach for renal fibrosis.

Comparison of anti-angiogenic properties of pristine carbon nanoparticles

DEFF Research Database (Denmark)

Wierzbicki, Mateusz; Sawosz, Ewa; Grodzik, Marta

2013-01-01

nanomaterials on blood vessel development. Diamond nanoparticles, graphite nanoparticles, graphene nanosheets, multi-wall nanotubes and C60 fullerenes were evaluated for their angiogenic activities using the in ovo chick embryo chorioallantoic membrane model. Diamond nanoparticles and multi-wall nanotubes...... showed the greatest anti-angiogenic properties. Interestingly, fullerene exhibited the opposite effect, increasing blood vessel development, while graphite nanoparticles and graphene had no effect. Subsequently, protein levels of pro-angiogenic growth factor receptors were analysed, showing that diamond...... nanoparticles decreased the expression of vascular endothelial growth factor receptor. These results provide new insights into the biological activity of carbon nanomaterials and emphasise the potential use of multi-wall nanotubes and diamond nanoparticles in anti-angiogenic tumour therapy....

Erkitinib, a novel EGFR tyrosine kinase inhibitor screened using a ProteoChip system from a phytochemical library

Energy Technology Data Exchange (ETDEWEB)

Kim, Eung-Yoon; Choi, Young-Jin [Biochip Research Center, Hoseo University, Asan 336-795 (Korea, Republic of); Innopharmascreen, Inc., Asan 336-795 (Korea, Republic of); Park, Chan-Won [Biochip Research Center, Hoseo University, Asan 336-795 (Korea, Republic of); Dept. of Biological Science, Hoseo University, Asan 336-795 (Korea, Republic of); Kang, In-Cheol, E-mail: ickang@hoseo.edu [Biochip Research Center, Hoseo University, Asan 336-795 (Korea, Republic of); Dept. of Biological Science, Hoseo University, Asan 336-795 (Korea, Republic of); Innopharmascreen, Inc., Asan 336-795 (Korea, Republic of)

2009-11-20

Receptor tyrosine kinases (PTKs) play key roles in the pathogenesis of numerous human diseases, including cancer. Therefore PTK inhibitors are currently under intensive investigation as potential drug candidates. Herein, we report on a ProteoChip-based screening of an epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitor, Erkitinibs, from phytochemical libraries. PLC-{gamma}-1 was used as a substrate immobilized on a ProteoChip and incubated with an EGFR kinase to phosphorylate tyrosine residues of the substrate, followed by a fluorescence detection of the substrate recognized by a phospho-specific monoclonal antibody. Erkitinibs inhibited HeLa cell proliferation in a dose-dependent manner. In conclusion, these data suggest that Erkitinibs can be a specific inhibitor of an EGFR kinase and can be further developed as a potent anti-tumor agent.

Erkitinib, a novel EGFR tyrosine kinase inhibitor screened using a ProteoChip system from a phytochemical library

International Nuclear Information System (INIS)

Kim, Eung-Yoon; Choi, Young-Jin; Park, Chan-Won; Kang, In-Cheol

2009-01-01

Receptor tyrosine kinases (PTKs) play key roles in the pathogenesis of numerous human diseases, including cancer. Therefore PTK inhibitors are currently under intensive investigation as potential drug candidates. Herein, we report on a ProteoChip-based screening of an epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitor, Erkitinibs, from phytochemical libraries. PLC-γ-1 was used as a substrate immobilized on a ProteoChip and incubated with an EGFR kinase to phosphorylate tyrosine residues of the substrate, followed by a fluorescence detection of the substrate recognized by a phospho-specific monoclonal antibody. Erkitinibs inhibited HeLa cell proliferation in a dose-dependent manner. In conclusion, these data suggest that Erkitinibs can be a specific inhibitor of an EGFR kinase and can be further developed as a potent anti-tumor agent.

Ibrutinib: a first in class covalent inhibitor of Bruton's tyrosine kinase.

Science.gov (United States)

Davids, Matthew S; Brown, Jennifer R

2014-05-01

Ibrutinib (formerly PCI-32765) is a potent, covalent inhibitor of Bruton's tyrosine kinase, a kinase downstream of the B-cell receptor that is critical for B-cell survival and proliferation. In preclinical studies, ibrutinib bound to Bruton's tyrosine kinase with high affinity, leading to inhibition of B-cell receptor signaling, decreased B-cell activation and induction of apoptosis. In clinical studies, ibrutinib has been well-tolerated and has demonstrated profound anti-tumor activity in a variety of hematologic malignancies, most notably chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), leading to US FDA approval for relapsed CLL and MCL. Ongoing studies are evaluating ibrutinib in other types of non-Hodgkin's lymphoma, such as diffuse large B-cell lymphoma and Waldenström's macrogobulinemia, in larger Phase III studies in CLL and MCL, and in combination studies with monoclonal antibodies and chemotherapy. Future studies will combine ibrutinib with other promising novel agents currently in development in hematologic malignancies.

Putative tyrosine kinases expressed in K-562 human leukemia cells

International Nuclear Information System (INIS)

Partanen, J.; Maekelae, T.P.; Lehvaeslaiho, H.; Alitalo, K.; Alitalo, R.

1990-01-01

Tyrosine phosphorylation is important in the transmission of growth and differentiation signals; known tyrosine kinases include several oncoproteins and growth factor receptors. Interestingly, some differentiated cell types, such as erythrocytes and platelets contain high amounts of phosphotyrosine. The authors analyzed tyrosine kinases expressed in the K-562 chronic myelogenous leukemia cell line, which has a bipotential erythroid and megakaryoblastoid differentiation capacity. Analysis of 359 polymerase chain reaction-amplified cDNA clones led to the identification of 14 different tyrosine kinase-related sequences (JTK1-14). Two of the clones (JTK2 and JTK4) represent unusual members of the fibroblast growth factor receptor gene family, and the clones JTK5, JTK11, and JTK14 may also belong to the family of receptor tyrosine kinases but lack a close relationship to any known tyrosine kinase. Each of these different genes has its own characteristic expression pattern in K-562 cells and several other human tumor cell lines. In addition, the JTK11 and JTK14 mRNAs are induced during the megakaryoblastoid differentiation of K-562 cells. These tyrosine kinases may have a role in the differentiation of megakaryoblasts or in the physiology of platelets

Src protein-tyrosine kinase structure and regulation

International Nuclear Information System (INIS)

Roskoski, Robert

2004-01-01

Src and Src-family protein kinases are proto-oncogenes that play key roles in cell morphology, motility, proliferation, and survival. v-Src (a viral protein) is encoded by the chicken oncogene of Rous sarcoma virus, and Src (the cellular homologue) is encoded by a physiological gene, the first of the proto-oncogenes. From the N- to C-terminus, Src contains an N-terminal 14-carbon myristoyl group, a unique segment, an SH3 domain, an SH2 domain, a protein-tyrosine kinase domain, and a C-terminal regulatory tail. The chief phosphorylation sites of Src include tyrosine 416 that results in activation from autophosphorylation and tyrosine 527 that results in inhibition from phosphorylation by C-terminal Src kinase. In the restrained state, the SH2 domain forms a salt bridge with phosphotyrosine 527, and the SH3 domain binds to the kinase domain via a polyproline type II left-handed helix. The SH2 and SH3 domains occur on the backside of the kinase domain away from the active site where they stabilize a dormant enzyme conformation. Protein-tyrosine phosphatases such as PTPα displace phosphotyrosine 527 from the Src SH2 domain and mediate its dephosphorylation leading to Src kinase activation. C-terminal Src kinase consists of an SH3, SH2, and kinase domain; it lacks an N-terminal myristoyl group and a C-terminal regulatory tail. Its X-ray structure has been determined, and the SH2 lobe occupies a position that is entirely different from that of Src. Unlike Src, the C-terminal Src kinase SH2 and SH3 domains stabilize an active enzyme conformation. Amino acid residues in the αD helix near the catalytic loop in the large lobe of C-terminal Src kinase serve as a docking site for the physiological substrate (Src) but not for an artificial substrate (polyGlu 4 Tyr)

Complexes of γ-tubulin with nonreceptor protein tyrosine kinases Src and Fyn in differentiating P19 embryonal carcinoma cells

International Nuclear Information System (INIS)

Kukharskyy, Vitaliy; Sulimenko, Vadym; Macurek, Libor; Sulimenko, Tetyana; Draberova, Eduarda; Draber, Pavel

2004-01-01

Nonreceptor protein tyrosine kinases of the Src family have been shown to play an important role in signal transduction as well as in regulation of microtubule protein interactions. Here we show that γ-tubulin (γ-Tb) in P19 embryonal carcinoma cells undergoing neuronal differentiation is phosphorylated and forms complexes with protein tyrosine kinases of the Src family, Src and Fyn. Elevated expression of both kinases during differentiation corresponded with increased level of proteins phosphorylated on tyrosine. Immunoprecipitation experiments with antibodies against Src, Fyn, γ-tubulin, and with anti-phosphotyrosine antibody revealed that γ-tubulin appeared in complexes with these kinases. In vitro kinase assays showed tyrosine phosphorylation of proteins in γ-tubulin complexes isolated from differentiated cells. Pretreatment of cells with Src family selective tyrosine kinase inhibitor PP2 reduced the amount of phosphorylated γ-tubulin in the complexes. Binding experiments with recombinant SH2 and SH3 domains of Src and Fyn kinases revealed that protein complexes containing γ-tubulin bound to SH2 domains and that these interactions were of SH2-phosphotyrosine type. The combined data suggest that Src family kinases might have an important role in the regulation of γ-tubulin interaction with tubulin dimers or other proteins during neurogenesis

The anti-proliferative and anti-angiogenic effect of the methanol extract from brittle star.

Science.gov (United States)

Baharara, Javad; Amini, Elaheh; Mousavi, Marzieh

2015-04-01

Anti-angiogenic therapy is a crucial step in cancer treatment. The discovery of new anti-angiogenic compounds from marine organisms has become an attractive concept in anti-cancer therapy. Because little data correlated to the pro- and anti-angiogenic efficacies of Ophiuroidea, which include brittle star, the current study was designed to explore the anti-angiogenic potential of brittle star methanol extract in vitro and in vivo. The anti-proliferative effect of brittle star extract on A2780cp cells was examined by MTT assays, and transcriptional expression of VEGF and b-FGF was evaluated by RT-PCR. In an in vivo model, 40 fertilized Ross eggs were divided into control and three experimental groups. The experimental groups were incubated with brittle star extract at concentrations of 25, 50 and 100 µg/ml, and photographed by photo-stereomicroscopy. Ultimately, numbers and lengths of vessels were measured by Image J software. Data were analyzed with SPSS software (pstar extract exerted a dose- and time-dependent anti-proliferative effect on A2780cp cancer cells. In addition, VEGF and b-FGF expression decreased with brittle star methanol extract treatment. Macroscopic evaluations revealed significant changes in the second and third experimental group compared to controls (pstar methanol extract in vitro and in vivo confer novel insight into the application of natural marine products in angiogenesis-related pathologies.

Smoking in preeclamptic women is associated with higher birthweight for gestational age and lower soluble fms-like tyrosine kinase-1 levels: a nested case control study

Directory of Open Access Journals (Sweden)

Kahn Susan R

2011-11-01

Full Text Available Abstract Background Smoking paradoxically increases the risk of small-for-gestational-age (SGA birth but protects against preeclampsia. Some studies have reported a "U-shaped" distribution of fetal growth in preeclamptic pregnancies, but reasons for this are unknown. We investigated whether cigarette smoking interacts with preeclampsia to affect fetal growth, and compared levels of soluble fms-like tyrosine kinase-1 (sFlt-1, a circulating anti-angiogenic protein, in preeclamptic smokers and non-smokers. Methods From a multicenter cohort of 5337 pregnant women, we prospectively identified 113 women who developed preeclampsia (cases and 443 controls. Smoking exposure was assessed by self-report and maternal hair nicotine levels. Fetal growth was assessed as z-score of birthweight for gestational age (BWGA. sFlt-1 was measured in plasma samples collected at the 24-26-week visit. Results In linear regression, smoking and preeclampsia were each associated with lower BWGA z-scores (β = -0.29; p = 0.008, and β = -0.67; p Conclusions Maternal smoking seems to protect against preeclampsia-associated fetal growth restriction and may account, at least partly, for the U-shaped pattern of fetal growth described in preeclamptic pregnancies. Smoking may exert this effect by reducing levels of the anti-angiogenic protein sFlt-1.

Ror receptor tyrosine kinases: orphans no more

OpenAIRE

Green, Jennifer L.; Kuntz, Steven G.; Sternberg, Paul W.

2008-01-01

Receptor tyrosine kinase-like orphan receptor (Ror) proteins are a conserved family of tyrosine kinase receptors that function in developmental processes including skeletal and neuronal development, cell movement and cell polarity. Although Ror proteins were originally named because the associated ligand and signaling pathway were unknown, recent studies in multiple species have now established that Ror proteins are Wnt receptors. Depending on the cellular context, Ror proteins can either act...

Anti-angiogenic activity of a new andrographolide derivative in zebrafish and HUVECs.

Science.gov (United States)

Li, Jingjing; Peng, Yuran; Li, Shang; Sun, Yicheng; Chan, Judy Yuet-Wa; Cui, Guozhen; Wang, Decai; Zhou, Guo-Chun; Lee, Simon Ming-Yuen

2016-10-15

Andrographolide is among the most promising anti-tumor and anti-angiogenic components in Andrographis paniculata but its poor bioavailability and limited efficacy pose difficulties for its therapeutic development. Therefore, improving its pharmaceutical features and potency, by modifying its chemical structure, is desirable. In the present study, a new andrographolide derivative (AGP-40) was synthesized and characterized for its anti-angiogenic properties. Human umbilical vein endothelial cells (HUVECs) and zebrafish models were used to identify the anti-angiogenic activity of AGP-40. AGP-40 significantly suppressed the formation of blood vessels in zebrafish and inhibited proliferation, migration and tube formation in vitro. The anti-angiogenic effects of AGP-40 are at least partially mediated via the PI3K/Akt and MEK/Erk(1/2) signaling pathways. Furthermore, AGP-40 exhibited stronger anti-proliferative effects than andrographolide against A549, HepG2, Hela cancer cell lines. This study is the first to demonstrate the promising anti-angiogenic activity of the new andrographolide derivative AGP-40. Our results indicate that AGP-40 could serve as a potential therapeutic agent for the treatment and prevention of diseases associated with excessive angiogenesis. Copyright © 2016 Elsevier B.V. All rights reserved.

Ibrutinib: a first in class covalent inhibitor of Bruton’s tyrosine kinase

Science.gov (United States)

Davids, Matthew S; Brown, Jennifer R

2015-01-01

Ibrutinib (formerly PCI-32765) is a potent, covalent inhibitor of Bruton’s tyrosine kinase, a kinase downstream of the B-cell receptor that is critical for B-cell survival and proliferation. In preclinical studies, ibrutinib bound to Bruton’s tyrosine kinase with high affinity, leading to inhibition of B-cell receptor signaling, decreased B-cell activation and induction of apoptosis. In clinical studies, ibrutinib has been well-tolerated and has demonstrated profound anti-tumor activity in a variety of hematologic malignancies, most notably chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), leading to US FDA approval for relapsed CLL and MCL. Ongoing studies are evaluating ibrutinib in other types of non-Hodgkin’s lymphoma, such as diffuse large B-cell lymphoma and Waldenström’s macrogobulinemia, in larger Phase III studies in CLL and MCL, and in combination studies with monoclonal antibodies and chemotherapy. Future studies will combine ibrutinib with other promising novel agents currently in development in hematologic malignancies. PMID:24941982

Anti-angiogenic activity and phytochemical screening of fruit fractions from Vitex agnus castus.

Science.gov (United States)

Certo, Giovanna; Costa, Rosaria; D'Angelo, Valeria; Russo, Marina; Albergamo, Ambrogina; Dugo, Giacomo; Germanò, Maria Paola

2017-12-01

Although the antitumour activity of Vitex agnus castus fruits has been already addressed, no work has yet assessed their anti-angiogenic potential. To this purpose, several extractive fractions of such fruits were tested on zebrafish embrios by EAP assay, so that only the bioactive fractions could be subsequently tested on the chick chorioallantoic membrane by CAM assay. Bioactive fractions were also phytochemically screened to identify those bioactive compounds responsible for anti-angiogenic activity. A marked inhibition of vessel formation was detected only in zebrafish embryos treated with chloroform or ethyl acetate fractions. Considering CAM assay, chloroform fraction induced a strong reduction of microvasculature and haemoglobin content; while lower anti-angiogenic effects of the ethyl acetate fraction were determined. Phytochemical analyses confirmed the presence of several bioactive anti-angiogenic compounds. Overall, obtained preliminary results highlighted a potential anti-angiogenic activity of V. agnus castus fruits.

Constitutive Activity in an Ancestral Form of Abl Tyrosine Kinase.

Directory of Open Access Journals (Sweden)

Saadat U Aleem

Full Text Available The c-abl proto-oncogene encodes a nonreceptor tyrosine kinase that is found in all metazoans, and is ubiquitously expressed in mammalian tissues. The Abl tyrosine kinase plays important roles in the regulation of mammalian cell physiology. Abl-like kinases have been identified in the genomes of unicellular choanoflagellates, the closest relatives to the Metazoa, and in related unicellular organisms. Here, we have carried out the first characterization of a premetazoan Abl kinase, MbAbl2, from the choanoflagellate Monosiga brevicollis. The enzyme possesses SH3, SH2, and kinase domains in a similar arrangement to its mammalian counterparts, and is an active tyrosine kinase. MbAbl2 lacks the N-terminal myristoylation and cap sequences that are critical regulators of mammalian Abl kinase activity, and we show that MbAbl2 is constitutively active. When expressed in mammalian cells, MbAbl2 strongly phosphorylates cellular proteins on tyrosine, and transforms cells much more potently than mammalian Abl kinase. Thus, MbAbl2 appears to lack the autoinhibitory mechanism that tightly constrains the activity of mammalian Abl kinases, suggesting that this regulatory apparatus arose more recently in metazoan evolution.

Importance of tyrosine phosphorylation in receptor kinase complexes.

Science.gov (United States)

Macho, Alberto P; Lozano-Durán, Rosa; Zipfel, Cyril

2015-05-01

Tyrosine phosphorylation is an important post-translational modification that is known to regulate receptor kinase (RK)-mediated signaling in animals. Plant RKs are annotated as serine/threonine kinases, but recent work has revealed that tyrosine phosphorylation is also crucial for the activation of RK-mediated signaling in plants. These initial observations have paved the way for subsequent detailed studies on the mechanism of activation of plant RKs and the biological relevance of tyrosine phosphorylation for plant growth and immunity. In this Opinion article we review recent reports on the contribution of RK tyrosine phosphorylation in plant growth and immunity; we propose that tyrosine phosphorylation plays a major regulatory role in the initiation and transduction of RK-mediated signaling in plants. Copyright © 2015 Elsevier Ltd. All rights reserved.

Anti-inflammatory and angiogenic activity of polysaccharide extract obtained from Tibetan kefir.

Science.gov (United States)

Prado, Maria Rosa Machado; Boller, Christian; Zibetti, Rosiane Guetter Mello; de Souza, Daiany; Pedroso, Luciana Lopes; Soccol, Carlos Ricardo

2016-11-01

The search for new bioactive molecules is a driving force for research pharmaceutical industries, especially those molecules obtained from fermentation. The molecules possessing angiogenic and anti-inflammatory attributes have attracted attention and are the focus of this study. Angiogenic activity from kefir polysaccharide extract, via chorioallantoic membrane assay, exhibited a pro-angiogenic effect compared with vascular endothelial factor (pro-angiogenic) and hydrocortisone (anti-angiogenic) activity as standards with an EC50 of 192ng/mL. In terms of anti-inflammatory activity determined via hyaluronidase enzyme assay, kefir polysaccharide extract inhibited the enzyme with a minimal activity of 2.08mg/mL and a maximum activity of 2.57mg/mL. For pharmaceutical purposes, kefir polysaccharide extract is considered to be safe because it does not inhibit VERO cells in cytotoxicity assays. Copyright © 2016 Elsevier Inc. All rights reserved.

Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells

International Nuclear Information System (INIS)

Okabe, Seiichi; Tauchi, Tetsuzo; Tanaka, Yuko; Ohyashiki, Kazuma

2013-01-01

Highlights: •Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells okabe et al. •Imatinib or nilotinib resistance was involved Src family kinase. •The BCR-ABL point mutation (E334V) was highly resistant to imatinib or nilotinib. •Ponatinib was a powerful strategy against imatinib or nilotinib resistant Ph-positive cells. -- Abstract: Because a substantial number of patients with chronic myeloid leukemia acquire resistance to ABL tyrosine kinase inhibitors (TKIs), their management remains a challenge. Ponatinib, also known as AP24534, is an oral multi-targeted TKI. Ponatinib is currently being investigated in a pivotal phase 2 clinical trial. In the present study, we analyzed the molecular and functional consequences of ponatinib against imatinib- or nilotinib-resistant (R) K562 and Ba/F3 cells. The proliferation of imatinib- or nilotinib-resistant K562 cells did not decrease after treatment with imatinib or nilotinib. Src family kinase Lyn was activated. Point mutation Ba/F3 cells (E334 V) were also highly resistant to imatinib and nilotinib. Treatment with ponatinib for 72 h inhibited the growth of imatinib- and nilotinib-resistant cells. The phosphorylation of BCR-ABL, Lyn, and Crk-L was reduced. This study demonstrates that ponatinib has an anti-leukemia effect by reducing ABL and Lyn kinase activity and this information may be of therapeutic relevance

Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells

Energy Technology Data Exchange (ETDEWEB)

Okabe, Seiichi, E-mail: okabe@tokyo-med.ac.jp; Tauchi, Tetsuzo; Tanaka, Yuko; Ohyashiki, Kazuma

2013-06-07

Highlights: •Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells okabe et al. •Imatinib or nilotinib resistance was involved Src family kinase. •The BCR-ABL point mutation (E334V) was highly resistant to imatinib or nilotinib. •Ponatinib was a powerful strategy against imatinib or nilotinib resistant Ph-positive cells. -- Abstract: Because a substantial number of patients with chronic myeloid leukemia acquire resistance to ABL tyrosine kinase inhibitors (TKIs), their management remains a challenge. Ponatinib, also known as AP24534, is an oral multi-targeted TKI. Ponatinib is currently being investigated in a pivotal phase 2 clinical trial. In the present study, we analyzed the molecular and functional consequences of ponatinib against imatinib- or nilotinib-resistant (R) K562 and Ba/F3 cells. The proliferation of imatinib- or nilotinib-resistant K562 cells did not decrease after treatment with imatinib or nilotinib. Src family kinase Lyn was activated. Point mutation Ba/F3 cells (E334 V) were also highly resistant to imatinib and nilotinib. Treatment with ponatinib for 72 h inhibited the growth of imatinib- and nilotinib-resistant cells. The phosphorylation of BCR-ABL, Lyn, and Crk-L was reduced. This study demonstrates that ponatinib has an anti-leukemia effect by reducing ABL and Lyn kinase activity and this information may be of therapeutic relevance.

All-trans-retinoic acid enhances apoptosis induction by tyrosine kinase inhibitors in the eosinophilic leukemia-derived EoL-1 cell line.

Science.gov (United States)

Robert, Carine; Apàti, Agota; Chomienne, Christine; Papp, Béla

2008-02-01

Imatinib and retinoids induce apoptosis in FIP1L1/PDGFRalpha-positive EoL-1 leukemia cells. Although imatinib induces complete remission in most FIP1L1/PDGFRalpha-positive patients, response to imatinib is sometimes suboptimal. In order to enhance the potency of the molecularly targeted therapy of eosinophilic leukemia, we investigated the effect of retinoids combined with tyrosine kinase inhibitors on EoL-1 cells. We demonstrate that retinoids combined with tyrosine kinase inhibitors lead to enhanced apoptosis induction in EoL-1 cells. Our results suggest that tyrosine kinase inhibitors combined with retinoids may constitute a valuable therapeutic approach for sensitive neoplasias that may display enhanced anti-leukemic potency when compared to single drug treatments.

SH2 domains: modulators of nonreceptor tyrosine kinase activity.

Science.gov (United States)

Filippakopoulos, Panagis; Müller, Susanne; Knapp, Stefan

2009-12-01

The Src homology 2 (SH2) domain is a sequence-specific phosphotyrosine-binding module present in many signaling molecules. In cytoplasmic tyrosine kinases, the SH2 domain is located N-terminally to the catalytic kinase domain (SH1) where it mediates cellular localization, substrate recruitment, and regulation of kinase activity. Initially, structural studies established a role of the SH2 domain stabilizing the inactive state of Src family members. However, biochemical characterization showed that the presence of the SH2 domain is frequently required for catalytic activity, suggesting a crucial function stabilizing the active state of many nonreceptor tyrosine kinases. Recently, the structure of the SH2-kinase domain of Fes revealed that the SH2 domain stabilizes the active kinase conformation by direct interactions with the regulatory helix alphaC. Stabilizing interactions between the SH2 and the kinase domains have also been observed in the structures of active Csk and Abl. Interestingly, mutations in the SH2 domain found in human disease can be explained by SH2 domain destabilization or incorrect positioning of the SH2. Here we summarize our understanding of mechanisms that lead to tyrosine kinase activation by direct interactions mediated by the SH2 domain and discuss how mutations in the SH2 domain trigger kinase inactivation.

Solubilization and characterization of a novel tyrosine kinase from rat adipocytes

International Nuclear Information System (INIS)

Yagaloff, K.A.; Czech, M.P.

1987-01-01

The authors report the efficient solubilization and characterization of a Triton X-100 insoluble tyrosine kinase from rat adipocytes. Plasma membranes were prepared from rat epididymal fat pads and were solubilized in 1% Triton X-100. Following centrifugation, the pellet was solubilized for 15 min at 4 0 C using both ionic and non-ionic detergents. Tyrosine kinase activity was measured in the soluble and particulate fractions using the exogenous substrate poly(glu-tyr) in a TCA precipitation assay. Reactions were performed in 50mM Hepes, 10mM MgCl 2 and 100μM gamma[ 32 P]-ATP (10Ci/mmol) at 4 0 C with or without 1mg/ml of the polyaminoacid. Incorporation rates of 100 to 1000 pmol/min/mg were obtained, while endogenous [ 32 P] incorporation was typically less than 10% of that in the presence of poly(glu-tyr). More than 75% of the tyrosine kinase activity was recovered in the soluble supernatant using this assay methodology. The solubilized tyrosine kinase was found to require Mg 2+ or Mn 2+ but preferred Mg 2+ and was inhibited by high levels of Mn 2+ . Kinase activity was strongly inhibited by Ca 2+ (>50% at 1mM), NaCl (>50% at 250mM) and NH 4 SO 4 (>50% at 50mM) but was activated by 10μM heparin and 5mM dithiothreitol. These properties distinguish the solubilized tyrosine kinase from other cellular tyrosine kinases

Involvement of the N-terminal unique domain of Chk tyrosine kinase in Chk-induced tyrosine phosphorylation in the nucleus

International Nuclear Information System (INIS)

Nakayama, Yuji; Kawana, Akiko; Igarashi, Asae; Yamaguchi, Naoto

2006-01-01

Chk tyrosine kinase phosphorylates Src-family kinases and suppresses their kinase activity. We recently showed that Chk localizes to the nucleus as well as the cytoplasm and inhibits cell proliferation. In this study, we explored the role of the N-terminal unique domain of Chk in nuclear localization and Chk-induced tyrosine phosphorylation in the nucleus. In situ binding experiments showed that the N-terminal domain of Chk was associated with the nucleus and the nuclear matrix. The presence of the N-terminal domain of Chk led to a fourfold increase in cell population exhibiting Chk-induced tyrosine phosphorylation in the nucleus. Expression of Chk but not kinase-deficient Chk induced tyrosine phosphorylation of a variety of proteins ranging from 23 kDa to ∼200 kDa, especially in Triton X-100-insoluble fraction that included chromatin and the nuclear matrix. Intriguingly, in situ subnuclear fractionations revealed that Chk induced tyrosine phosphorylation of proteins that were associated with the nuclear matrix. These results suggest that various unidentified substrates of Chk, besides Src-family kinases, may be present in the nucleus. Thus, our findings indicate that the importance of the N-terminal domain to Chk-induced tyrosine phosphorylation in the nucleus, implicating that these nuclear tyrosine-phosphorylated proteins may contribute to inhibition of cell proliferation

Antibody-induced activation of the epidermal growth factor receptor tyrosine kinase requires the presence of detergent

NARCIS (Netherlands)

Spaargaren, M.; Defize, L. H.; de Laat, S. W.; Boonstra, J.

1990-01-01

Activation of the epidermal growth factor receptor (EGF-R) tyrosine kinase was investigated in membrane preparations as well as intact A431 cells, using anti-EGF-R antibodies directed against extra- and intracellular receptor domains. In vitro assay conditions were mimicked on whole cells by a mild

Expression Profiling of Tyrosine Kinase Genes

National Research Council Canada - National Science Library

Weier, Heinz

2000-01-01

... of these genes parallels the progression of tumors to a more malignant phenotype. We developed a DNA micro-array based screening system to monitor the level of expression of tyrosine kinase (tk...

In vitro and in vivo anti-angiogenic activities of Panduratin A.

Directory of Open Access Journals (Sweden)

Siew-Li Lai

Full Text Available Targeting angiogenesis has emerged as an attractive and promising strategy in anti-cancer therapeutic development. The present study investigates the anti-angiogenic potential of Panduratin A (PA, a natural chalcone isolated from Boesenbergia rotunda by using both in vitro and in vivo assays.PA exerted selective cytotoxicity on human umbilical vein endothelial cells (HUVECs with IC(50 value of 6.91 ± 0.85 µM when compared to human normal fibroblast and normal liver epithelial cells. Assessment of the growth kinetics by cell impedance-based Real-Time Cell Analyzer showed that PA induced both cytotoxic and cytostatic effects on HUVECs, depending on the concentration used. Results also showed that PA suppressed VEGF-induced survival and proliferation of HUVECs. Furthermore, endothelial cell migration, invasion, and morphogenesis or tube formation demonstrated significant time- and dose-dependent inhibition by PA. PA also suppressed matrix metalloproteinase-2 (MMP-2 secretion and attenuated its activation to intermediate and active MMP-2. In addition, PA suppressed F-actin stress fiber formation to prevent migration of the endothelial cells. More importantly, anti-angiogenic potential of PA was also evidenced in two in vivo models. PA inhibited neo-vessels formation in murine Matrigel plugs, and angiogenesis in zebrafish embryos.Taken together, our study demonstrated the distinctive anti-angiogenic properties of PA, both in vitro and in vivo. This report thus reveals another biological activity of PA in addition to its reported anti-inflammatory and anti-cancer activities, suggestive of PA's potential for development as an anti-angiogenic agent for cancer therapy.

VEGFR tyrosine kinase inhibitor II (VRI) induced vascular insufficiency in zebrafish as a model for studying vascular toxicity and vascular preservation

International Nuclear Information System (INIS)

Li, Shang; Dang, Yuan Ye; Oi Lam Che, Ginny; Kwan, Yiu Wa; Chan, Shun Wan; Leung, George Pak Heng; Lee, Simon Ming Yuen; Hoi, Maggie Pui Man

2014-01-01

In ischemic disorders such as chronic wounds and myocardial ischemia, there is inadequate tissue perfusion due to vascular insufficiency. Besides, it has been observed that prolonged use of anti-angiogenic agents in cancer therapy produces cardiovascular toxicity caused by impaired vessel integrity and regeneration. In the present study, we used VEGFR tyrosine kinase inhibitor II (VRI) to chemically induce vascular insufficiency in zebrafish in vivo and human umbilical vein endothelial cells (HUVEC) in vitro to further study the mechanisms of vascular morphogenesis in these pathological conditions. We also explored the possibility of treating vascular insufficiency by enhancing vascular regeneration and repair with pharmacological intervention. We observed that pretreatment of VRI induced blood vessel loss in developing zebrafish by inhibiting angiogenesis and increasing endothelial cell apoptosis, accompanied by down-regulation of kdr, kdrl and flt-1 genes expression. The VRI-induced blood vessel loss in zebrafish could be restored by post-treatment of calycosin, a cardiovascular protective isoflavone. Similarly, VRI induced cytotoxicity and apoptosis in HUVEC which could be rescued by calycosin post-treatment. Further investigation of the underlying mechanisms showed that the PI3K/AKT/Bad cell survival pathway was a main contributor of the vascular regenerative effect of calycosin. These findings indicated that the cardiovascular toxicity in anti-angiogenic therapy was mainly caused by insufficient endothelial cell survival, suggesting its essential role in vascular integrity, repair and regeneration. In addition, we showed that VRI-induced blood vessel loss in zebrafish represented a simple and effective in vivo model for studying vascular insufficiency and evaluating cancer drug vascular toxicities. - Highlights: • In vivo VRI model • Rescue effects of calycosin • Calycosin EC survival pathways

Novel Bruton's tyrosine kinase inhibitors currently in development

Directory of Open Access Journals (Sweden)

D'Cruz OJ

2013-03-01

Full Text Available Osmond J D'Cruz,1 Fatih M Uckun1,21Children's Center for Cancer and Blood Diseases, Children's Hospital Los Angeles, Los Angeles, CA, USA; 2Department of Pediatrics, University of Southern California, Los Angeles, CA, USAAbstract: Bruton's tyrosine kinase (Btk is intimately involved in multiple signal-transduction pathways regulating survival, activation, proliferation, and differentiation of B-lineage lymphoid cells. Btk is overexpressed and constitutively active in several B-lineage lymphoid malignancies. Btk has emerged as a new antiapoptotic molecular target for treatment of B-lineage leukemias and lymphomas. Preclinical and early clinical results indicate that Btk inhibitors may be useful in the treatment of leukemias and lymphomas.Keywords: tyrosine kinase, personalized therapy, kinase inhibitors, Btk, leukemia, lymphoma

Tyrosine Kinase Receptor Landscape in Lung Cancer: Therapeutical Implications

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A. Quintanal-Villalonga

2016-01-01

Full Text Available Lung cancer is a heterogeneous disease responsible for the most cases of cancer-related deaths. The majority of patients are clinically diagnosed at advanced stages, with a poor survival rate. For this reason, the identification of oncodrivers and novel biomarkers is decisive for the future clinical management of this pathology. The rise of high throughput technologies popularly referred to as “omics” has accelerated the discovery of new biomarkers and drivers for this pathology. Within them, tyrosine kinase receptors (TKRs have proven to be of importance as diagnostic, prognostic, and predictive tools and, due to their molecular nature, as therapeutic targets. Along this review, the role of TKRs in the different lung cancer histologies, research on improvement of anti-TKR therapy, and the current approaches to manage anti-TKR resistance will be discussed.

Identification and analysis of a novel protein-tyrosine kinase from bovine thymus

International Nuclear Information System (INIS)

Zioncheck, T.F.; Harrison, M.L.; Geahlen, R.L.

1986-01-01

A cytosolic protein-tyrosine kinase has been identified and purified to near homogeneity from calf thymus by using the phosphorylation of the tyrosine-containing peptide angiotensin I as an assay. Specific peptide phosphorylating activity was enhanced by carrying out the assay at high ionic strength (2M NaCl). The inclusion of NaCl at this concentration acts to stimulate endogenous protein-tyrosine kinase activity while simultaneously inhibiting other endogenous kinases. The purification procedure involved extraction of the enzyme from calf-thymus and sequential chromatography on columns of DEAE-cellulose, heparin-agarose, casein-sepharose, butylagarose, and Sephadex G-75. Analysis of the most highly purified preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single Coomassie blue-stained band of 41 KDa. This molecular weight was consistent with results obtained from gel filtration, indicating that the enzyme exists as a monomer. The enzyme has also been found to catalyze an autophosphorylation reaction. Incubation of the enzyme with Mn 2+ and [γ- 32 P]ATP led to its modification on a tyrosine residue. Phosphopeptide mapping experiments indicated that the 41 KDa kinase was distinct from p56, the major membrane-associated protein-tyrosine kinase in T lymphocytes

Requirement for tyrosine phosphatase during serotonergic neuromodulation by protein kinase C.

Science.gov (United States)

Catarsi, S; Drapeau, P

1997-08-01

Tyrosine kinases and phosphatases are abundant in the nervous system, where they signal cellular differentiation, mediate the responses to growth factors, and direct neurite outgrowth during development. Tyrosine phosphorylation can also alter ion channel activity, but its physiological significance remains unclear. In an identified leech mechanosensory neuron, the ubiquitous neuromodulator serotonin increases the activity of a cation channel by activating protein kinase C (PKC), resulting in membrane depolarization and modulation of the receptive field properties. We observed that the effects on isolated neurons and channels were blocked by inhibiting tyrosine phosphatases. Serotonergic stimulation of PKC thus activates a tyrosine phosphatase activity associated with the channels, which reverses their constitutive inhibition by tyrosine phosphorylation, representing a novel form of neuromodulation.

Receptor tyrosine kinase signaling: a view from quantitative proteomics

DEFF Research Database (Denmark)

Dengjel, Joern; Kratchmarova, Irina; Blagoev, Blagoy

2009-01-01

Growth factor receptor signaling via receptor tyrosine kinases (RTKs) is one of the basic cellular communication principals found in all metazoans. Extracellular signals are transferred via membrane spanning receptors into the cytoplasm, reversible tyrosine phosphorylation being the hallmark of all...

Pervanadate induces Mammalian Ste20 Kinase 3 (MST3) tyrosine phosphorylation but not activation.

Science.gov (United States)

Kan, Wei-Chih; Lu, Te-Ling; Ling, Pin; Lee, Te-Hsiu; Cho, Chien-Yu; Huang, Chi-Ying F; Jeng, Wen-Yih; Weng, Yui-Ping; Chiang, Chun-Yen; Wu, Jin Bin; Lu, Te-Jung

2016-07-01

The yeast Ste20 (sterile) protein kinase, which is a serine/threonine kinase, responds to the stimulation of the G proteincoupled receptor (GPCR) pheromone receptor. Ste20 protein kinase serves as the critical component that links signaling from the GPCR/G proteins to the mitogen-activated protein kinase (MAPK) cascade in yeast. The yeast Ste20p functions as a MAP kinase kinase kinase kinase (MAP4K) in the pheromone response. Ste20-like kinases are structurally conserved from yeast to mammals. The mechanism by which MAP4K links GPCR to the MAPK pathway is less clearly defined in vertebrates. In addition to MAP4K, the tyrosine kinase cascade bridges G proteins and the MAPK pathway in vertebrate cells. Mammalian Ste20 Kinase 3 (MST3) has been categorized into the Ste20 family and has been reported to function in the regulation of cell polarity and migration. However, whether MST3 tyrosine phosphorylation regulates diverse signaling pathways is unknown. In this study, the tyrosine phosphatase inhibitor pervanadate was found to induce MST3 tyrosine phosphorylation in intact cells, and the activity of tyrosine-phosphorylated MST3 was measured. This tyrosine-directed phosphorylation was independent of MST3 activity. Parameters including protein conformation, Triton concentration and ionic concentration influenced the sensitivity of MST3 activity. Taken together, our data suggests that the serine/threonine kinase MST3 undergoes tyrosinedirected phosphorylation. The tyrosine-phosphorylated MST3 may create a docking site for the structurally conserved SH2/SH3 (Src Homology 2 and 3) domains within the Src oncoprotein. The unusual tyrosinephosphorylated MST3 may recruit MST3 to various signaling components. Copyright © 2016. Published by Elsevier Inc.

Evidence for association of the cloned liver growth hormone receptor with a tyrosine kinase

DEFF Research Database (Denmark)

Wang, X; Uhler, M D; Billestrup, N

1992-01-01

The ability of the cloned liver growth hormone (GH) receptor, when expressed in mammalian cell lines, to copurify with tyrosine kinase activity and be tyrosyl phosphorylated was examined. 125I-human growth hormone-GH receptor complexes isolated from COS-7 cells transiently expressing high levels...... of tyrosine kinase activity with cloned liver GH receptor. The level of phosphorylation of the GH receptor was very low, as compared with the endogenous GH receptor in 3T3-F442A cells, suggesting that tyrosine kinase activity is not intrinsic to the cloned GH receptor but rather resides with a kinase present...... in a variety of cell types. The finding that the level of phosphorylation of GH receptor appears to vary with cell type is consistent with the cloned liver GH receptor being a substrate for an associated tyrosine kinase and with the amount of such a GH receptor-associated tyrosine kinase being cell type-specific....

Ror receptor tyrosine kinases: orphans no more.

Science.gov (United States)

Green, Jennifer L; Kuntz, Steven G; Sternberg, Paul W

2008-11-01

Receptor tyrosine kinase-like orphan receptor (Ror) proteins are a conserved family of tyrosine kinase receptors that function in developmental processes including skeletal and neuronal development, cell movement and cell polarity. Although Ror proteins were originally named because the associated ligand and signaling pathway were unknown, recent studies in multiple species have now established that Ror proteins are Wnt receptors. Depending on the cellular context, Ror proteins can either activate or repress transcription of Wnt target genes and can modulate Wnt signaling by sequestering Wnt ligands. New evidence implicates Ror proteins in planar cell polarity, an alternative Wnt pathway. Here, we review the progress made in understanding these mysterious proteins and, in particular, we focus on their function as Wnt receptors.

SOCS proteins in regulation of receptor tyrosine kinase signaling

DEFF Research Database (Denmark)

Kazi, Julhash U.; Kabir, Nuzhat N.; Flores Morales, Amilcar

2014-01-01

Receptor tyrosine kinases (RTKs) are a family of cell surface receptors that play critical roles in signal transduction from extracellular stimuli. Many in this family of kinases are overexpressed or mutated in human malignancies and thus became an attractive drug target for cancer treatment....... The signaling mediated by RTKs must be tightly regulated by interacting proteins including protein-tyrosine phosphatases and ubiquitin ligases. The suppressors of cytokine signaling (SOCS) family proteins are well-known negative regulators of cytokine receptors signaling consisting of eight structurally similar...

Tyrosine Kinase Ligand-Receptor Pair Prediction by Using Support Vector Machine

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Masayuki Yarimizu

2015-01-01

Full Text Available Receptor tyrosine kinases are essential proteins involved in cellular differentiation and proliferation in vivo and are heavily involved in allergic diseases, diabetes, and onset/proliferation of cancerous cells. Identifying the interacting partner of this protein, a growth factor ligand, will provide a deeper understanding of cellular proliferation/differentiation and other cell processes. In this study, we developed a method for predicting tyrosine kinase ligand-receptor pairs from their amino acid sequences. We collected tyrosine kinase ligand-receptor pairs from the Database of Interacting Proteins (DIP and UniProtKB, filtered them by removing sequence redundancy, and used them as a dataset for machine learning and assessment of predictive performance. Our prediction method is based on support vector machines (SVMs, and we evaluated several input features suitable for tyrosine kinase for machine learning and compared and analyzed the results. Using sequence pattern information and domain information extracted from sequences as input features, we obtained 0.996 of the area under the receiver operating characteristic curve. This accuracy is higher than that obtained from general protein-protein interaction pair predictions.

Tyrosine kinase fusion genes in pediatric BCR-ABL1-like acute lymphoblastic leukemia

Science.gov (United States)

Boer, Judith M.; Steeghs, Elisabeth M.P.; Marchante, João R.M.; Boeree, Aurélie; Beaudoin, James J.; Berna Beverloo, H.; Kuiper, Roland P.; Escherich, Gabriele; van der Velden, Vincent H.J.; van der Schoot, C. Ellen; de Groot-Kruseman, Hester A.; Pieters, Rob; den Boer, Monique L.

2017-01-01

Approximately 15% of pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL) is characterized by gene expression similar to that of BCR-ABL1-positive disease and unfavorable prognosis. This BCR-ABL1-like subtype shows a high frequency of B-cell development gene aberrations and tyrosine kinase-activating lesions. To evaluate the clinical significance of tyrosine kinase gene fusions in children with BCP-ALL, we studied the frequency of recently identified tyrosine kinase fusions, associated genetic features, and prognosis in a representative Dutch/German cohort. We identified 14 tyrosine kinase fusions among 77 BCR-ABL1-like cases (18%) and none among 76 non-BCR-ABL1-like B-other cases. Novel exon fusions were identified for RCSD1-ABL2 and TERF2-JAK2. JAK2 mutation was mutually exclusive with tyrosine kinase fusions and only occurred in cases with high CRLF2 expression. The non/late response rate and levels of minimal residual disease in the fusion-positive BCR-ABL1-like group were higher than in the non-BCR-ABL1-like B-others (p<0.01), and also higher, albeit not statistically significant, compared with the fusion-negative BCR-ABL1-like group. The 8-year cumulative incidence of relapse in the fusion-positive BCR-ABL1-like group (35%) was comparable with that in the fusion-negative BCR-ABL1-like group (35%), and worse than in the non-BCR-ABL1-like B-other group (17%, p=0.07). IKZF1 deletions, predominantly other than the dominant-negative isoform and full deletion, co-occurred with tyrosine kinase fusions. This study shows that tyrosine kinase fusion-positive cases are a high-risk subtype of BCP-ALL, which warrants further studies with specific kinase inhibitors to improve outcome. PMID:27894077

Tyrosine kinase signalling in breast cancer

International Nuclear Information System (INIS)

Hynes, Nancy E

2000-01-01

Cells are continuously exposed to diverse stimuli ranging from soluble endocrine and paracrine factors to signalling molecules on neighbouring cells. Receptors of the tyrosine kinase family play an important role in the integration and interpretation of these external stimuli, allowing a cell to respond appropriately to its environment. The activation of receptor tyrosine kinases (RTKs) is tightly controlled, allowing a normal cell to correctly integrate its external environment with internal signal transduction pathways. In contrast, due to numerous molecular alterations arising during the course of malignancy, a tumour is characterized by an abnormal response to its environment, which allows cancer cells to evade the normal mechanisms controlling cellular proliferation. Alterations in the expression of various RTKs, in their activation, and in the signalling molecules lying downstream of the receptors play important roles in the development of cancer. This topic is the major focus of the thematic review section of this issue of Breast Cancer Research

PTB domain-directed substrate targeting in a tyrosine kinase from the unicellular choanoflagellate Monosiga brevicollis.

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Victoria Prieto-Echagüe

2011-04-01

Full Text Available Choanoflagellates are considered to be the closest living unicellular relatives of metazoans. The genome of the choanoflagellate Monosiga brevicollis contains a surprisingly high number and diversity of tyrosine kinases, tyrosine phosphatases, and phosphotyrosine-binding domains. Many of the tyrosine kinases possess combinations of domains that have not been observed in any multicellular organism. The role of these protein interaction domains in M. brevicollis kinase signaling is not clear. Here, we have carried out a biochemical characterization of Monosiga HMTK1, a protein containing a putative PTB domain linked to a tyrosine kinase catalytic domain. We cloned, expressed, and purified HMTK1, and we demonstrated that it possesses tyrosine kinase activity. We used immobilized peptide arrays to define a preferred ligand for the third PTB domain of HMTK1. Peptide sequences containing this ligand sequence are phosphorylated efficiently by recombinant HMTK1, suggesting that the PTB domain of HMTK1 has a role in substrate recognition analogous to the SH2 and SH3 domains of mammalian Src family kinases. We suggest that the substrate recruitment function of the noncatalytic domains of tyrosine kinases arose before their roles in autoinhibition.

Ethyl p-methoxycinnamate from Kaempferia galanga inhibits angiogenesis through tyrosine kinase

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Juni Ekowati

2015-04-01

Full Text Available Background Many tumors express on their receptor tyrosine kinases vascular endothelial growth factor activity associated with angiogenesis. Inhibition of angiogenesis through reduction of tyrosine kinase activity is a promising strategy for cancer therapy. The present study aimed to determine the mechanism and potency of ethyl p-methoxycinnamate (EPMC isolated from Kaempferia galanga as angiogenesis inhibitor. Methods A laboratory experimental study was conducted using chorio-allantoic membranes (CAMs of nine-day old chicken eggs induced by 60ng basic fibroblast growth factor (bFGF. Ethyl p-methoxycinnamate (EPMC potency was determined at dosages of 30, 60, 90 and 120 mg and compared with celecoxib 60 mg as reference drug and one negative bFGF-induced control group. Neovascularization and endothelial cell count in CAM blood vessels were evaluated. To predict the antiangiogenic mechanism of EPMC, a docking study was performed with the Molegro Virtual Docker program on tyrosine kinase as receptor (PDB 1XKK. Results Angiogenesis stimulation by bFGF was prevented significantly (p<0.05 by EPMC at dosages of 30, 60, 90 and 120 mg and this activity was dose dependent. Molecular docking showed interaction between EPMC functional groups and tyrosine kinase amino acids at Met766, Met793, Thr854, Thr790, Gln791 and Ala743. There was an association between EPMC antiangiogenic activity and docking study results. Conclusions Ethyl p-methoxycinnamate is a potential new angiogenesis inhibitor through interaction with tyrosine kinase. EPMC could be a promising therapeutic agent for treatment of angiogenesis-related diseases.

Perforated Gastric Ulcer Associated with Anti-Angiogenic Therapy

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Diogo Libânio

2017-08-01

Full Text Available Anti-angiogenic therapy with bevacizumab, an inhibitor of vascular endothelial growth factor, is commonly used in metastatic colorectal cancer and is rarely associated with gastrointestinal perforation, perforation being more frequent in the primary tumor site or at the anastomotic level. We present the case of a 64-year-old male with stage IV rectal adenocarcinoma who was on palliative chemotherapy with FOLFOX and bevacizumab. After the 4th chemotherapy cycle, our patient started fever and epigastric pain. He was hemodynamically stable, and signs of peritoneal irritation were absent. There were no alterations in the abdominal X-ray, and C-reactive protein was markedly elevated. A CT scan revealed a de novo thickness in the gastric antrum. Upper digestive endoscopy showed an ulcerated 40-mm lesion in the angulus, with a 20-mm orifice communicating with an exsudative cavity revested by the omentum. A conservative approach was decided including fasting, broad-spectrum intravenous antibiotics, and proton-pump inhibitors. Subsequent gastroduodenal series showed no contrast extravasation, allowing the resumption of oral nutrition. Esophagogastroduodenoscopy after 8 weeks showed perforation closure. Biopsies did not show neoplastic cells or Heliobacter pylori infection. Although the success in the conservative management of perforation allowing the maintenance of palliative chemotherapy (without bevacizumab, the patient died after 4 months due to liver failure. The reported case shows an uncommon endoscopic finding due to a rare complication of anti-angiogenic therapy. Additionally, it reminds clinicians that a history of gastroduodenal ulcers should be actively sought before starting anti-angiogenic treatment and that suspicion for perforation should be high in these cases.

SH2/SH3 adaptor proteins can link tyrosine kinases to a Ste20-related protein kinase, HPK1.

Science.gov (United States)

Anafi, M; Kiefer, F; Gish, G D; Mbamalu, G; Iscove, N N; Pawson, T

1997-10-31

Ste20-related protein kinases have been implicated as regulating a range of cellular responses, including stress-activated protein kinase pathways and the control of cytoskeletal architecture. An important issue involves the identities of the upstream signals and regulators that might control the biological functions of mammalian Ste20-related protein kinases. HPK1 is a protein-serine/threonine kinase that possesses a Ste20-like kinase domain, and in transfected cells activates a protein kinase pathway leading to the stress-activated protein kinase SAPK/JNK. Here we have investigated candidate upstream regulators that might interact with HPK1. HPK1 possesses an N-terminal catalytic domain and an extended C-terminal tail with four proline-rich motifs. The SH3 domains of Grb2 bound in vitro to specific proline-rich motifs in the HPK1 tail and functioned synergistically to direct the stable binding of Grb2 to HPK1 in transfected Cos1 cells. Epidermal growth factor (EGF) stimulation did not affect the binding of Grb2 to HPK1 but induced recruitment of the Grb2.HPK1 complex to the autophosphorylated EGF receptor and to the Shc docking protein. Several activated receptor and cytoplasmic tyrosine kinases, including the EGF receptor, stimulated the tyrosine phosphorylation of the HPK1 serine/threonine kinase. These results suggest that HPK1, a mammalian Ste20-related protein-serine/threonine kinase, can potentially associate with protein-tyrosine kinases through interactions mediated by SH2/SH3 adaptors such as Grb2. Such interaction may provide a possible mechanism for cross-talk between distinct biochemical pathways following the activation of tyrosine kinases.

Second-generation inhibitors of Bruton tyrosine kinase

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Jingjing Wu

2016-09-01

Full Text Available Abstract Bruton tyrosine kinase (BTK is a critical effector molecule for B cell development and plays a major role in lymphoma genesis. Ibrutinib is the first-generation BTK inhibitor. Ibrutinib has off-target effects on EGFR, ITK, and Tec family kinases, which explains the untoward effects of ibrutinib. Resistance to ibrutinib was also reported. The C481S mutation in the BTK kinase domain was reported to be a major mechanism of resistance to ibrutinib. This review summarizes the clinical development of novel BTK inhibitors, ACP-196 (acalabrutinib, ONO/GS-4059, and BGB-3111.

Structural basis for the regulation mechanism of the tyrosine kinase CapB from Staphylococcus aureus.

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Vanesa Olivares-Illana

2008-06-01

Full Text Available Bacteria were thought to be devoid of tyrosine-phosphorylating enzymes. However, several tyrosine kinases without similarity to their eukaryotic counterparts have recently been identified in bacteria. They are involved in many physiological processes, but their accurate functions remain poorly understood due to slow progress in their structural characterization. They have been best characterized as copolymerases involved in the synthesis and export of extracellular polysaccharides. These compounds play critical roles in the virulence of pathogenic bacteria, and bacterial tyrosine kinases can thus be considered as potential therapeutic targets. Here, we present the crystal structures of the phosphorylated and unphosphorylated states of the tyrosine kinase CapB from the human pathogen Staphylococcus aureus together with the activator domain of its cognate transmembrane modulator CapA. This first high-resolution structure of a bacterial tyrosine kinase reveals a 230-kDa ring-shaped octamer that dissociates upon intermolecular autophosphorylation. These observations provide a molecular basis for the regulation mechanism of the bacterial tyrosine kinases and give insights into their copolymerase function.

Protein kinase D1 signaling in angiogenic gene expression and VEGF-mediated angiogenesis

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Bin eRen MD, Phd, FAHA

2016-05-01

Full Text Available Protein kinase D 1 (PKD-1 is a signaling kinase important in fundamental cell functions including migration, proliferation and differentiation. PKD-1 is also a key regulator of gene expression and angiogenesis that is essential for cardiovascular development and tumor progression. Further understanding molecular aspects of PKD-1 signaling in the regulation of angiogenesis may have translational implications in obesity, cardiovascular disease and cancer. The author will summarize and provide the insights into molecular mechanisms by which PKD-1 regulates transcriptional expression of angiogenic genes, focusing on the transcriptional regulation of CD36 by PKD-1-FoxO1 signaling axis along with the potential implications of this axis in arterial differentiation and morphogenesis. He will also discuss a new concept of dynamic balance between proangiogenic and antiangiogenic signaling in determining angiogenic switch, and stress how PKD-1 signaling regulates VEGF signaling-mediated angiogenesis.

Tyrosine Kinase Gene Expression Profiling in Prostate Cancer

National Research Council Canada - National Science Library

Weier, Heinz-Ulrich

2001-01-01

... of these genes parallels the progression of tumors to a more malignant phenotype. We developed a DNA micro-array based screening system to monitor the level of expression of tyrosine kinase (tk...

Tyrosine Kinase Gene Expression Profiling in Prostate Cancer

National Research Council Canada - National Science Library

Weier, Heinz-Ulrich

2002-01-01

... of these genes parallels the progression of tumors to a more malignant phenotype. We developed a DNA micro-array based screening system to monitor the level of expression of tyrosine kinase (tk...

Non-invasive imaging for studying anti-angiogenic therapy effects

NARCIS (Netherlands)

Ehling, J.; Lammers, Twan Gerardus Gertudis Maria; Kiessling, F.

2013-01-01

Noninvasive imaging plays an emerging role in preclinical and clinical cancer research and has high potential to improve clinical translation of new drugs. This article summarises and discusses tools and methods to image tumour angiogenesis and monitor anti-angiogenic therapy effects. In this

Tyrosine phosphorylation of Grb14 by Tie2

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Dumont Daniel J

2010-10-01

Full Text Available Abstract Background Growth factor receptor bound (Grb proteins 7, 10 and 14 are a family of structurally related multi-domain adaptor proteins involved in a variety of biological processes. Grb7, 10 and 14 are known to become serine and/or threonine phosphorylated in response to growth factor (GF stimulation. Grb7 and 10 have also been shown to become tyrosine phosphorylated under certain conditions. Under experimental conditions Grb7 is tyrosine phosphorylated by the Tie2/Tie-2/Tek angiogenic receptor tyrosine kinase (RTK. Furthermore, Grb14 has also been shown to interact with Tie2, however tyrosine phosphorylation of this Grb family member has yet to be reported. Results Here we report for the first time tyrosine phosphorylation of Grb14. This phosphorylation requires a kinase competent Tie2 as well as intact tyrosines 1100 and 1106 (Y1100 and Y1106 on the receptor. Furthermore, a complete SH2 domain on Grb14 is required for Grb14 tyrosine phosphorylation by Tie2. Grb14 was also able to become tyrosine phosphorylated in primary endothelial cells when treated with a soluble and potent variant of the Tie2 ligand, cartilage oligomeric matrix protein (COMP Ang1. Conclusion Our results show that Grb14, like its family members Grb7 and Grb10, is able to be tyrosine phosphorylated. Furthermore, our data indicate a role for Grb14 in endothelial signaling downstream of the Tie2 receptor.

Muscle-Specific Tyrosine Kinase and Myasthenia Gravis Owing to Other Antibodies.

Science.gov (United States)

Rivner, Michael H; Pasnoor, Mamatha; Dimachkie, Mazen M; Barohn, Richard J; Mei, Lin

2018-05-01

Around 20% of patients with myasthenia gravis are acetylcholine receptor antibody negative; muscle-specific tyrosine kinase antibodies (MuSK) were identified as the cause of myasthenia gravis in 30% to 40% of these cases. Anti MuSK myasthenia gravis is associated with specific clinical phenotypes. One is a bulbar form with fewer ocular symptoms. Others show an isolated head drop or symptoms indistinguishable from acetylcholine receptor-positive myasthenia gravis. These patients usually respond well to immunosuppressive therapy, but not as well to cholinesterase inhibitors. Other antibodies associated with myasthenia gravis, including low-density lipoprotein receptor-related protein 4, are discussed. Copyright © 2018 Elsevier Inc. All rights reserved.

Giant hub Src and Syk tyrosine kinase thermodynamic profiles recapitulate evolution

Science.gov (United States)

Phillips, J. C.

2017-10-01

Thermodynamic scaling theory, previously applied mainly to small proteins, here analyzes quantitative evolution of the titled functional network giant hub enzymes. The broad domain structure identified homologically is confirmed hydropathically using amino acid sequences only. The most surprising results concern the evolution of the tyrosine kinase globular surface roughness from avians to mammals, which is first order, compared to the evolution within mammals from rodents to humans, which is second order. The mystery of the unique amide terminal region of proto oncogene tyrosine protein kinase is resolved by the discovery there of a rare hydroneutral septad targeting cluster, which is paralleled by an equally rare octad catalytic cluster in tyrosine kinase in humans and a few other species (cat and dog). These results, which go far towards explaining why these proteins are among the largest giant hubs in protein interaction networks, use no adjustable parameters.

Tyrosine kinase inhibitors for brain metastases in HER2-positive breast cancer.

Science.gov (United States)

Duchnowska, Renata; Loibl, Sibylle; Jassem, Jacek

2018-06-01

Approximately 30-50% of advanced HER2-positive breast cancer patients will develop central nervous system (CNS) metastases, with an annual risk of around 10%, and a half of them will die from brain progression. An increased risk of brain metastases is also seen in patients with early HER2-positive breast cancer administered curative therapy. Brain metastases in HER2-positive breast cancer patients usually constitute the first site of recurrence. The administration of anti-HER2 monoclonal antibodies, trastuzumab and pertuzumab, considerably delays the onset of symptomatic brain disease: however, the limited penetration of these compounds into the CNS hinders their efficacy. The small-molecule tyrosine kinase inhibitors of epidermal growth factor receptors family have established activity in HER2-positive breast cancer in both advanced disease and neoadjuvant setting. Favorable physico-chemical properties of these compounds allow them for a more efficient penetration through the blood-brain barrier, and hold the promise for more effective prevention and treatment of brain metastases. In this article we review the role of currently available or investigational HER2 tyrosine kinase inhibitors: lapatinib, neratinib, afatinib and tucatinib in the treatment of brain metastases in HER2-positive breast cancer patients. Copyright © 2018 Elsevier Ltd. All rights reserved.

SH2 domains: modulators of nonreceptor tyrosine kinase activity

OpenAIRE

Filippakopoulos, Panagis; Müller, Susanne; Knapp, Stefan

2009-01-01

The Src homology 2 (SH2) domain is a sequence-specific phosphotyrosine-binding module present in many signaling molecules. In cytoplasmic tyrosine kinases, the SH2 domain is located N-terminally to the catalytic kinase domain (SH1) where it mediates cellular localization, substrate recruitment, and regulation of kinase activity. Initially, structural studies established a role of the SH2 domain stabilizing the inactive state of Src family members. However, biochemical characterization showed ...

Defibrotide: an endothelium protecting and stabilizing drug, has an anti-angiogenic potential in vitro and in vivo.

Science.gov (United States)

Koehl, Gudrun E; Geissler, Edward K; Iacobelli, Massimo; Frei, Caroline; Burger, Verena; Haffner, Silvia; Holler, Ernst; Andreesen, Reinhard; Schlitt, Hans J; Eissner, Günther

2007-05-01

Defibrotide (DF) is a polydisperse mixture of 90% single-stranded oligonucleotides with anti-thrombotic and anti-apoptotic functions. DF is used in the treatment of endothelial complications in the course of allogeneic stem cell transplantation. Recent preclinical evidence suggests that DF might also have anti-neoplastic properties. In the present study we hypothesized that DF might inhibit tumors via an anti-angiogenic effect. The anti-angiogenic potential of DF was tested in vitro using human microvascular endothelial cells forming vessel structures across a layer of dermal fibroblasts. Our results show that pharmacologic DF concentrations (100 mug/ml) significantly reduced vessel formation in this assay. Similarly, DF blocked sprouting from cultured rat aortic rings. In vivo, angiogenesis in a human gastric tumor (TMK1) implanted in dorsal skin-fold chambers (in nude mice) was inhibited by i.v. application of 450 mg/kg DF. Notably, due to its short half-life, DF was most effective when given on a daily basis. Although the precise mechanism of DF remains to be elucidated, initial Western blots show that DF reduces phosphorylation-activation of p70S6 kinase, which is a key target in the PI3K/Akt/mTOR signaling pathway linked to endothelial cell and pericyte proliferation and activation. However, in vitro data suggest that DF acts independently of vascular endothelial growth factor. Taken together, our data suggest that while DF is known for its endothelium-protecting function in SCT, it also inhibits formation of new blood vessels, and thus should be considered for further testing as an adjuvant anti-cancer agent, either alone, or in combination with other drugs.

Biomarkers for Anti-Angiogenic Therapy in Cancer

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Markus Wehland

2013-04-01

Full Text Available Angiogenesis, the development of new vessels from existing vasculature, plays a central role in tumor growth, survival, and progression. On the molecular level it is controlled by a number of pro- and anti-angiogenic cytokines, among which the vascular endothelial growth factors (VEGFs, together with their related VEGF-receptors, have an exceptional position. Therefore, the blockade of VEGF signaling in order to inhibit angiogenesis was deemed an attractive approach for cancer therapy and drugs interfering with the VEGF-ligands, the VEGF receptors, and the intracellular VEGF-mediated signal transduction were developed. Although promising in pre-clinical trials, VEGF-inhibition proved to be problematic in the clinical context. One major drawback was the generally high variability in patient response to anti-angiogenic drugs and the rapid development of therapy resistance, so that, in total, only moderate effects on progression-free and overall survival were observed. Biomarkers predicting the response to VEGF-inhibition might attenuate this problem and help to further individualize drug and dosage determination. Although up to now no definitive biomarker has been identified for this purpose, several candidates are currently under investigation. This review aims to give an overview of the recent developments in this field, focusing on the most prevalent tumor species.

Celecoxib restores angiogenic factor expression at the maternal-fetal interface in the BPH/5 mouse model of preeclampsia.

Science.gov (United States)

Reijnders, Dorien; Liu, Chin-Chi; Xu, Xinjing; Zhao, Anna M; Olson, Kelsey N; Butler, Scott D; Douglas, Nataki C; Sones, Jenny L

2018-05-01

Preeclampsia (PE), a hypertensive disease of pregnancy, is a leading cause of fetal and maternal morbidity/mortality. Early angiogenic and inflammatory disturbances within the placenta are thought to underlie the development of the maternal PE syndrome and poor pregnancy outcomes. However, the exact etiology remains largely unknown. Here, we use the BPH/5 mouse model of PE to elucidate the way in which inflammation early in pregnancy contributes to abnormal expression of angiogenic factors at the maternal-fetal interface. We have previously described improvement in maternal hypertension and fetal growth restriction in this model after treatment with the anti-inflammatory cyclooxygenase-2 (Cox2) specific inhibitor celecoxib. To further characterize the mechanisms by which celecoxib improves poor pregnancy outcomes in BPH/5 mice, we determined expression of angiogenic factors and complement pathway components after celecoxib. In BPH/5 implantation sites there was increased hypoxia inducible factor-1α ( Hif1α), heme oxygenase-1 ( Ho-1), and stem cell factor ( Scf) mRNA concomitant with elevated prostaglandin synthase 2 ( Ptgs2), encoding Cox2, and elevated VEGF protein. Angiopoietin 1 ( Ang1), tunica interna endothelial cell kinase-2 receptor ( Tie2), complement factor 3 ( C3), and complement factor B ( CfB) were increased in midgestation BPH/5 placentae. Whereas BPH/5 expression levels of VEGF, Ang1, and Tie2 normalized after celecoxib, placental C3 and CfB mRNA remained unchanged. However, celecoxib did reduce the pregnancy-specific circulating soluble fms-like tyrosine kinase-1 (sFlt-1) rise in BPH/5 mice at midgestation. These data show that elevated Cox2 during implantation contributes to placental angiogenic factor imbalances in the BPH/5 mouse model of PE.

Role of Bruton's tyrosine kinase in B cells and malignancies

NARCIS (Netherlands)

Pal Singh, S. (Simar); F. Dammeijer (Floris); R.W. Hendriks (Rudi)

2018-01-01

textabstractBruton's tyrosine kinase (BTK) is a non-receptor kinase that plays a crucial role in oncogenic signaling that is critical for proliferation and survival of leukemic cells in many B cell malignancies. BTK was initially shown to be defective in the primary immunodeficiency X-linked

Visceral fat area predicts survival in patients with advanced hepatocellular carcinoma treated with tyrosine kinase inhibitors.

Science.gov (United States)

Nault, Jean-Charles; Pigneur, Frédéric; Nelson, Anaïs Charles; Costentin, Charlotte; Tselikas, Lambros; Katsahian, Sandrine; Diao, Guoqing; Laurent, Alexis; Mallat, Ariane; Duvoux, Christophe; Luciani, Alain; Decaens, Thomas

2015-10-01

Anthropometric measurements have been linked to resistance to anti-angiogenic treatment and survival. Patients with advanced hepatocellular carcinoma treated with sorafenib or brivanib in 2008-2011 were included in this retrospective study. Anthropometric measurements were assessed using computed tomography and were correlated with drug toxicity, radiological response, and overall survival. 52 patients were included, Barcelona Clinic Liver Classification B (38%) and C (62%), with a mean value of α-fetoprotein of 29,554±85,654 ng/mL, with a median overall survival of 10.5 months. Sarcopenia was associated with a greater rate of hand-foot syndrome (P=0.049). Modified Response Evaluation Criteria In Solid Tumours (mRECIST) and Choi criteria were significantly associated with survival, but RECIST criteria were not. An absence of hand-foot syndrome and high-visceral fat area were associated with progressive disease as assessed by RECIST and mRECIST criteria. In multivariate analyses, high visceral fat area (HR=3.6; P=0.002), low lean body mass (HR=2.4; P=0.015), and presence of hand-foot syndrome (HR=1.8; P=0.004) were significantly associated with overall survival. In time-dependent multivariate analyses; only high visceral fat area was associated with survival. Visceral fat area is associated with survival and seems to be a predictive marker for primary resistance to tyrosine kinase inhibitors in patients with advanced hepatocellular carcinoma. Copyright © 2015 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

DMPD: Macrophage-stimulating protein and RON receptor tyrosine kinase: potentialregulators of macrophage inflammatory activities. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 12472665 Macrophage-stimulating protein and RON receptor tyrosine kinase: potential...:545-53. (.png) (.svg) (.html) (.csml) Show Macrophage-stimulating protein and RON receptor tyrosine kinase:...le Macrophage-stimulating protein and RON receptor tyrosine kinase: potentialregulators of macrophage inflam

Exploring the Hypersensitivity of PTEN Deleted Prostate Cancer Stem Cells to WEE1 Tyrosine Kinase Inhibitors

Science.gov (United States)

2015-12-01

AWARD NUMBER: W81XWH-14-1-0251 TITLE: Exploring the Hypersensitivity of PTEN Deleted Prostate Cancer Stem Cells to WEE1 Tyrosine Kinase... Tyrosine Kinase Inhibitors 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-14-1-0251 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Kiran Mahajan 5d...ABSTRACT Central to all cycling cells-including prostate cancer stem cells- is the expression of WEE1 tyrosine kinase. WEE1 monitors duplication of

Oncogenic Receptor Tyrosine Kinases Directly Phosphorylate Focal Adhesion Kinase (FAK) as a Resistance Mechanism to FAK-Kinase Inhibitors.

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Marlowe, Timothy A; Lenzo, Felicia L; Figel, Sheila A; Grapes, Abigail T; Cance, William G

2016-12-01

Focal adhesion kinase (FAK) is a major drug target in cancer and current inhibitors targeted to the ATP-binding pocket of the kinase domain have entered clinical trials. However, preliminary results have shown limited single-agent efficacy in patients. Despite these unfavorable data, the molecular mechanisms that drive intrinsic and acquired resistance to FAK-kinase inhibitors are largely unknown. We have demonstrated that receptor tyrosine kinases (RTK) can directly bypass FAK-kinase inhibition in cancer cells through phosphorylation of FAK's critical tyrosine 397 (Y397). We also showed that HER2 forms a direct protein-protein interaction with the FAK-FERM-F1 lobe, promoting direct phosphorylation of Y397. In addition, FAK-kinase inhibition induced two forms of compensatory RTK reprogramming: (i) the rapid phosphorylation and activation of RTK signaling pathways in RTK High cells and (ii) the long-term acquisition of RTKs novel to the parental cell line in RTK Low cells. Finally, HER2 +: cancer cells displayed resistance to FAK-kinase inhibition in 3D growth assays using a HER2 isogenic system and HER2 + cancer cell lines. Our data indicate a novel drug resistance mechanism to FAK-kinase inhibitors whereby HER2 and other RTKs can rescue and maintain FAK activation (pY397) even in the presence of FAK-kinase inhibition. These data may have important ramifications for existing clinical trials of FAK inhibitors and suggest that individual tumor stratification by RTK expression would be important to predict patient response to FAK-kinase inhibitors. Mol Cancer Ther; 15(12); 3028-39. ©2016 AACR. ©2016 American Association for Cancer Research.

Excretion of anti-angiogenic proteins in patients with chronic allograft dysfunction.

Science.gov (United States)

Moskowitz-Kassai, Eliza; Mackelaite, Lina; Chen, Jun; Patel, Kaushal; Dadhania, Darshana M; Gross, Steven S; Chander, Praveen; Delaney, Vera; Deng, Luqin; Chen, Ligong; Cui, Xiangqin; Suthanthiran, Manikkam; Goligorsky, Michael S

2012-02-01

We have recently documented the appearance of an anti-angiogenic peptide, endorepellin, in the urine of patients with chronic allograft dysfunction (CAD). Here, we analyzed using enzyme-linked immunosorbent assay the excretion of anti-angiogenic peptides endostatin, pigment epithelium-derived factor (PEDF) and Kruppel-like factor-2 (KLF-2), in healthy individuals, patients with stable graft function and patients with various degrees of CAD. In healthy subjects and patients with CAD-0, endostatin, PEDF and KLF-2 excretions were at the level of detection. In contrast, there were significant differences between the patients with CAD-3 and CAD-0, CAD-1 and healthy controls for endostatin and CAD-0 versus CAD-3 for PEDF, but no differences in KLF-2 excretion. Receiver operating characteristic (ROC) curve analyses demonstrated a highly discriminative profile for all three biomarkers: the combination of these parameters offered 83% sensitivity and 90% specificity in distinguishing CAD-0 from CAD-1-3. The quality of these potential biomarkers of CAD was, however, highest in discriminating CAD status in biopsy-proven cases and dropped when CAD-0 was diagnosed based on clinical criteria. In conclusion, these findings indicate the diagnostic potential of urinary detection of endostatin, PEDF and to lesser degree KLF-2 and suggest a mechanistic role played by anti-angiogenic substances in the developing vasculopathy and vascular rarefaction in patients with CAD.

BCR-ABL1 tyrosine kinase inhibitors for the treatment of chronic myeloid leukemia.

Science.gov (United States)

Cuellar, Sandra; Vozniak, Michael; Rhodes, Jill; Forcello, Nicholas; Olszta, Daniel

2017-01-01

The management of chronic myeloid leukemia with BCR-ABL1 tyrosine kinase inhibitors has evolved chronic myeloid leukemia into a chronic, manageable disease. A patient-centered approach is important for the appropriate management of chronic myeloid leukemia and optimization of long-term treatment outcomes. The pharmacist plays a key role in treatment selection, monitoring drug-drug interactions, identification and management of adverse events, and educating patients on adherence. The combination of tyrosine kinase inhibitors with unique safety profiles and individual patients with unique medical histories can make managing treatment difficult. This review will provide up-to-date information regarding tyrosine kinase inhibitor-based treatment of patients with chronic myeloid leukemia. Management strategies for adverse events and considerations for drug-drug interactions will not only vary among patients but also across tyrosine kinase inhibitors. Drug-drug interactions can be mild to severe. In instances where co-administration of concomitant medications cannot be avoided, it is critical to understand how drug levels are impacted and how subsequent dose modifications ensure therapeutic drug levels are maintained. An important component of patient-centered management of chronic myeloid leukemia also includes educating patients on the significance of early and regular monitoring of therapeutic milestones, emphasizing the importance of adhering to treatment in achieving these targets, and appropriately modifying treatment if these clinical goals are not being met. Overall, staying apprised of current research, utilizing the close pharmacist-patient relationship, and having regular interactions with patients, will help achieve successful long-term treatment of chronic myeloid leukemia in the age of BCR-ABL1 tyrosine kinase inhibitors.

Chimeric Mouse model to track the migration of bone marrow derived cells in glioblastoma following anti-angiogenic treatments.

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Achyut, B R; Shankar, Adarsh; Iskander, A S M; Ara, Roxan; Knight, Robert A; Scicli, Alfonso G; Arbab, Ali S

2016-01-01

Bone marrow derived cells (BMDCs) have been shown to contribute in the tumor development. In vivo animal models to investigate the role of BMDCs in tumor development are poorly explored. We established a novel chimeric mouse model using as low as 5 × 10(6) GFP+ BM cells in athymic nude mice, which resulted in >70% engraftment within 14 d. In addition, chimera was established in NOD-SCID mice, which displayed >70% with in 28 d. Since anti-angiogenic therapies (AAT) were used as an adjuvant against VEGF-VEGFR pathway to normalize blood vessels in glioblastoma (GBM), which resulted into marked hypoxia and recruited BMDCs to the tumor microenvironment (TME). We exploited chimeric mice in athymic nude background to develop orthotopic U251 tumor and tested receptor tyrosine kinase inhibitors and CXCR4 antagonist against GBM. We were able to track GFP+ BMDCs in the tumor brain using highly sensitive multispectral optical imaging instrument. Increased tumor growth associated with the infiltration of GFP+ BMDCs acquiring suppressive myeloid and endothelial phenotypes was seen in TME following treatments. Immunofluorescence study showed GFP+ cells accumulated at the site of VEGF, SDF1 and PDGF expression, and at the periphery of the tumors following treatments. In conclusion, we developed a preclinical chimeric model of GBM and phenotypes of tumor infiltrated BMDCs were investigated in context of AATs. Chimeric mouse model could be used to study detailed cellular and molecular mechanisms of interaction of BMDCs and TME in cancer.

Structural basis for the regulation mechanism of the tyrosine kinase CapB from Staphylococcus aureus

DEFF Research Database (Denmark)

Olivares-Illana, Vanesa; Meyer, Philippe; Bechet, Emmanuelle

2008-01-01

Bacteria were thought to be devoid of tyrosine-phosphorylating enzymes. However, several tyrosine kinases without similarity to their eukaryotic counterparts have recently been identified in bacteria. They are involved in many physiological processes, but their accurate functions remain poorly...... understood due to slow progress in their structural characterization. They have been best characterized as copolymerases involved in the synthesis and export of extracellular polysaccharides. These compounds play critical roles in the virulence of pathogenic bacteria, and bacterial tyrosine kinases can thus...... be considered as potential therapeutic targets. Here, we present the crystal structures of the phosphorylated and unphosphorylated states of the tyrosine kinase CapB from the human pathogen Staphylococcus aureus together with the activator domain of its cognate transmembrane modulator CapA. This first high...

Receptor protein tyrosine phosphatase alpha activates Src-family kinases and controls integrin-mediated responses in fibroblasts

DEFF Research Database (Denmark)

Su, J; Muranjan, M; Sap, J

1999-01-01

of tyrosine kinases, the activity of which is tightly controlled by inhibitory phosphorylation of a carboxyterminal tyrosine residue (Tyr527 in chicken c-Src); this phosphorylation induces the kinases to form an inactive conformation. Whereas the identity of such inhibitory Tyr527 kinases has been well...... established, no corresponding phosphatases have been identified that, under physiological conditions, function as positive regulators of c-Src and Fyn in fibroblasts. RESULTS: Receptor protein tyrosine phosphatase alpha (RPTPalpha) was inactivated by homologous recombination. Fibroblasts derived from...... these RPTPalpha-/- mice had impaired tyrosine kinase activity of both c-Src and Fyn, and this was accompanied by a concomitant increase in c-Src Tyr527 phosphorylation. RPTPalpha-/- fibroblasts also showed a reduction in the rate of spreading on fibronectin substrates, a trait that is a phenocopy of the effect...

Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues

Directory of Open Access Journals (Sweden)

Lei eShi

2014-09-01

Full Text Available Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells.

Dynamics of tumor growth and combination of anti-angiogenic and cytotoxic therapies

Science.gov (United States)

Kohandel, M.; Kardar, M.; Milosevic, M.; Sivaloganathan, S.

2007-07-01

Tumors cannot grow beyond a certain size (about 1-2 mm in diameter) through simple diffusion of oxygen and other essential nutrients into the tumor. Angiogenesis, the formation of blood vessels from pre-existing vessels, is a crucial and observed step, through which a tumor obtains its own blood supply. Thus, strategies that interfere with the development of this tumor vasculature, known as anti-angiogenic therapy, represent a novel approach to controlling tumor growth. Several pre-clinical studies have suggested that currently available angiogenesis inhibitors are unlikely to yield significant sustained improvements in tumor control on their own, but rather will need to be used in combination with conventional treatments to achieve maximal benefit. Optimal sequencing of anti-angiogenic treatment and radiotherapy or chemotherapy is essential to the success of these combined treatment strategies. Hence, a major challenge to mathematical modeling and computer simulations is to find appropriate dosages, schedules and sequencing of combination therapies to control or eliminate tumor growth. Here, we present a mathematical model that incorporates tumor cells and the vascular network, as well as their interplay. We can then include the effects of two different treatments, conventional cytotoxic therapy and anti-angiogenic therapy. The results are compared with available experimental and clinical data.

MHC-I-induced apoptosis in human B-lymphoma cells is dependent on protein tyrosine and serine/threonine kinases

DEFF Research Database (Denmark)

Pedersen, Anders Elm; Bregenholt, S; Johansen, B

1999-01-01

B lymphoma cells, is dependent on protein tyrosine kinases and the phosphatidylinositol 3 (PI-3) kinase. Functional studies showed that MHC-I crosslinking induced almost complete inhibition of the spontaneous proliferation of the B lymphoma cells as early as 6 h post-crosslinking and apoptosis 24 h...... post-crosslinking. Preincubation with either protein tyrosine kinase or protein serine/threonine kinase inhibitors reduced the MHC-I-induced apoptosis to background levels, whereas inhibition of PI-3 kinase had no effect. These data demonstrate a pivotal role for protein tyrosine and serine...

Oxidized LDL-induced angiogenesis involves sphingosine 1-phosphate: prevention by anti-S1P antibody.

Science.gov (United States)

Camaré, Caroline; Trayssac, Magali; Garmy-Susini, Barbara; Mucher, Elodie; Sabbadini, Roger; Salvayre, Robert; Negre-Salvayre, Anne

2015-01-01

Neovascularization occurring in atherosclerotic lesions may promote plaque expansion, intraplaque haemorrhage and rupture. Oxidized LDL (oxLDL) are atherogenic, but their angiogenic effect is controversial; both angiogenic and anti-angiogenic effects have been reported. The angiogenic mechanism of oxLDL is partly understood, but the role of the angiogenic sphingolipid, sphingosine 1-phosphate (S1P), in this process is not known. Thus, we investigated whether S1P is involved in the oxLDL-induced angiogenesis and whether an anti-S1P monoclonal antibody can prevent this effect. Angiogenesis was assessed by capillary tube formation by human microvascular endothelial cells (HMEC-1) cultured on Matrigel and in vivo by the Matrigel plug assay in C57BL/6 mice. Human oxLDL exhibited a biphasic angiogenic effect on HMEC-1; low concentrations were angiogenic, higher concentrations were cytotoxic. The angiogenic response to oxLDL was blocked by the sphingosine kinase (SPHK) inhibitor, dimethylsphingosine, by SPHK1-siRNA and by an anti-S1P monoclonal antibody. Moreover, inhibition of oxLDL uptake and subsequent redox signalling by anti-CD36 and anti-LOX-1 receptor antibodies and by N-acetylcysteine, respectively, blocked SPHK1 activation and tube formation. In vivo, in the Matrigel plug assay, low concentrations of human oxLDL or murine oxVLDL also triggered angiogenesis, which was prevented by i.p. injection of the anti-S1P antibody. These data highlight the role of S1P in angiogenesis induced by oxLDL both in HMEC-1 cultured on Matrigel and in vivo in the Matrigel plug model in mice, and demonstrate that the anti-S1P antibody effectively blocks the angiogenic effect of oxLDL. © 2014 The British Pharmacological Society.

Tyrosine phosphorylation in signal transduction

International Nuclear Information System (INIS)

Roberts, T.M.; Kaplan, D.; Morgan, W.; Keller, T.; Mamon, H.; Piwnica-Worms, H.; Druker, B.; Whitman, M.; Morrison, D.; Cohen, B.; Schaffhausen, B.; Cantley, L.; Rapp, U.

1988-01-01

Recent work has focused on the elucidation of the mechanisms by which membrane-bound tyrosine kinases transmit signals within the cell. To examine the role of tyrosine phosphorylation the authors have employed the following strategy. First, they have utilized antibodies to phosphotyrosine (anti-P.Tyr) to identify candidate substrates of various tyrosine kinases, such as pp60 c-src , the CSF- receptor, or the platelet-derived growth factor (PDGF) receptor. Second, they have attempted to characterize the biochemical properties of the putative substrates and to determine in what manner these properties are modified by phosphorylation on tyrosine residues. In this endeavor, they are recapitulating the classic biochemical analysis used to study the effect of kinases on metabolism. The final portion of our work consists of using modern molecular biological strategies to clone the genes or cDNAs for the substrates and overproduce the relevant proteins for studies in vitro in defined systems. This paper describes the first and second aspects of this strategy, the identification and characterization of novel substrate molecules

Protein tyrosine kinase and mitogen-activated protein kinase signalling pathways contribute to differences in heterophil-mediated innate immune responsiveness between two lines of broilers

Science.gov (United States)

Protein tyrosine phosphorylation mediates signal transduction of cellular processes, with protein tyrosine kinases (PTKs) regulating virtually all signaling events. The mitogen-activated protein kinase (MAPK) super-family consists of three conserved pathways that convert receptor activation into ce...

Rapid phospho-turnover by receptor tyrosine kinases impacts downstream signaling and drug binding.

Science.gov (United States)

Kleiman, Laura B; Maiwald, Thomas; Conzelmann, Holger; Lauffenburger, Douglas A; Sorger, Peter K

2011-09-02

Epidermal growth factor receptors (ErbB1-4) are oncogenic receptor tyrosine kinases (RTKs) that regulate diverse cellular processes. In this study, we combine measurement and mathematical modeling to quantify phospho-turnover at ErbB receptors in human cells and to determine the consequences for signaling and drug binding. We find that phosphotyrosine residues on ErbB1 have half-lives of a few seconds and therefore turn over 100-1000 times in the course of a typical immediate-early response to ligand. Rapid phospho-turnover is also observed for EGF-activated ErbB2 and ErbB3, unrelated RTKs, and multiple intracellular adaptor proteins and signaling kinases. Thus, the complexes formed on the cytoplasmic tail of active receptors and the downstream signaling kinases they control are highly dynamic and antagonized by potent phosphatases. We develop a kinetic scheme for binding of anti-ErbB1 drugs to receptors and show that rapid phospho-turnover significantly impacts their mechanisms of action. Copyright © 2011 Elsevier Inc. All rights reserved.

Autoinhibition of Bruton's tyrosine kinase (Btk) and activation by soluble inositol hexakisphosphate

Science.gov (United States)

Wang, Qi; Vogan, Erik M; Nocka, Laura M; Rosen, Connor E; Zorn, Julie A; Harrison, Stephen C; Kuriyan, John

2015-01-01

Bruton's tyrosine kinase (Btk), a Tec-family tyrosine kinase, is essential for B-cell function. We present crystallographic and biochemical analyses of Btk, which together reveal molecular details of its autoinhibition and activation. Autoinhibited Btk adopts a compact conformation like that of inactive c-Src and c-Abl. A lipid-binding PH-TH module, unique to Tec kinases, acts in conjunction with the SH2 and SH3 domains to stabilize the inactive conformation. In addition to the expected activation of Btk by membranes containing phosphatidylinositol triphosphate (PIP3), we found that inositol hexakisphosphate (IP6), a soluble signaling molecule found in both animal and plant cells, also activates Btk. This activation is a consequence of a transient PH-TH dimerization induced by IP6, which promotes transphosphorylation of the kinase domains. Sequence comparisons with other Tec-family kinases suggest that activation by IP6 is unique to Btk. DOI: http://dx.doi.org/10.7554/eLife.06074.001 PMID:25699547

Bosutinib induced pleural effusions: Case report and review of tyrosine kinase inhibitors induced pulmonary toxicity

Directory of Open Access Journals (Sweden)

Natalia I. Moguillansky, MD

2017-01-01

Full Text Available Tyrosine kinase inhibitors are known to cause pulmonary complications. We report a case of bosutinib related bilateral pleural effusions in a patient with chronic myeloid leukemia. Characteristics of the pleural fluid are presented. We also discuss other tyrosine kinase inhibitors induced pulmonary toxicities, including pulmonary hypertension and interstitial lung disease.

Predictive biomarker discovery through the parallel integration of clinical trial and functional genomics datasets

DEFF Research Database (Denmark)

Swanton, C.; Larkin, J.M.; Gerlinger, M.

2010-01-01

-cancer agents. The consortium focuses on the identification of reliable predictive biomarkers to approved agents with anti-angiogenic activity for which no reliable predictive biomarkers exist: sunitinib, a multi-targeted tyrosine kinase inhibitor and everolimus, a mammalian target of rapamycin (mTOR) pathway...

Circulating Angiogenic Factors and the Risk of Adverse Outcomes among Haitian Women with Preeclampsia.

Directory of Open Access Journals (Sweden)

Melissa I March

Full Text Available Angiogenic factors are strongly associated with adverse maternal and fetal outcomes among women with preterm preeclampsia (PE in developed countries. We evaluated the role of angiogenic factors and their relationship to adverse outcomes among Haitian women with PE.We measured plasma antiangiogenic soluble fms-like tyrosine kinase 1 (sFlt1 and proangiogenic placental growth factor (PlGF levels in women with PE (n=35 compared to controls with no hypertensive disorders (NHD (n=43 among subjects with singleton pregnancies that delivered at Hospital Albert Schweitzer (HAS in Haiti. We divided the preeclamptic women into two groups, early onset (≤ 34 weeks and late onset (>34 weeks and examined relationships between sFlt1/PlGF ratios on admission and adverse outcomes (abruption, respiratory complications, stroke, renal insufficiency, eclampsia, maternal death, birth weight 34 weeks with no adverse outcome.PE-related adverse outcomes are common in women in Haiti and are associated with profound angiogenic imbalance regardless of gestational age at presentation.

Regulation of hematopoietic cell function by protein tyrosine kinase-encoding oncogenes, a review

NARCIS (Netherlands)

Punt, C. J.

1992-01-01

Tyrosine phosphorylation of proteins by protein tyrosine kinases (PTKs) is an important mechanism in the regulation of various cellular processes such as proliferation, differentiation, and transformation. Accumulating data implicate PTKs as essential intermediates in the transduction of

Clinical Implication of Anti-Angiogenic Effect of Regorafenib in Metastatic Colorectal Cancer.

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Yoojoo Lim

Full Text Available Regorafenib induces distinct radiological changes that represent its anti-angiogenic effect. However, clinical implication of the changes is unclear.Tumor attenuation as measured by Hounsfield units (HU in contrast-enhanced computed tomography (CT and cavitary changes of lung metastases were analyzed in association with treatment outcome of metastatic colorectal cancer patients (N = 80 treated with regorafenib in a prospective study.141 lesions in 72 patients were analyzed with HU. After 2 cycles of regorafenib, 87.5% of patients showed decrease of HU (Median change -23.9%, range -61.5%-20.7%. Lesional attenuation change was modestly associated with metabolic changes of 18-fluoro-deoxyglucose positron emission tomography-CT (Pearson's r = 0.37, p = 0.002. Among 53 patients with lung metastases, 17 (32.1% developed cavitary changes. There were no differences in disease control rate, progression-free survival, or overall survival according to the radiological changes. At the time of progressive disease (PD according to RECIST 1.1, HU was lower than baseline in 86.0% (43/50 and cavitary change of lung metastasis persisted without refilling in 84.6% (11/13.Regorafenib showed prominent anti-angiogenic effect in colorectal cancer, but the changes were not associated with treatment outcome. However, the anti-angiogenic effects persisted at the time of PD, which suggests that we may need to develop new treatment strategies.

Clinical Implication of Anti-Angiogenic Effect of Regorafenib in Metastatic Colorectal Cancer

Science.gov (United States)

Yoon, Jeong Hee; Lee, Jeong Min; Lee, Jung Min; Paeng, Jin Chul; Won, Jae-Kyung; Kang, Gyeong Hoon; Jeong, Seung-Yong; Park, Kyu Joo; Lee, Kyung-Hun; Kim, Jee Hyun; Kim, Tae-You

2015-01-01

Background Regorafenib induces distinct radiological changes that represent its anti-angiogenic effect. However, clinical implication of the changes is unclear. Methods Tumor attenuation as measured by Hounsfield units (HU) in contrast-enhanced computed tomography (CT) and cavitary changes of lung metastases were analyzed in association with treatment outcome of metastatic colorectal cancer patients (N = 80) treated with regorafenib in a prospective study. Results 141 lesions in 72 patients were analyzed with HU. After 2 cycles of regorafenib, 87.5% of patients showed decrease of HU (Median change -23.9%, range -61.5%–20.7%). Lesional attenuation change was modestly associated with metabolic changes of 18-fluoro-deoxyglucose positron emission tomography-CT (Pearson’s r = 0.37, p = 0.002). Among 53 patients with lung metastases, 17 (32.1%) developed cavitary changes. There were no differences in disease control rate, progression-free survival, or overall survival according to the radiological changes. At the time of progressive disease (PD) according to RECIST 1.1, HU was lower than baseline in 86.0% (43/50) and cavitary change of lung metastasis persisted without refilling in 84.6% (11/13). Conclusion Regorafenib showed prominent anti-angiogenic effect in colorectal cancer, but the changes were not associated with treatment outcome. However, the anti-angiogenic effects persisted at the time of PD, which suggests that we may need to develop new treatment strategies. PMID:26671465

Function of Bruton's tyrosine kinase during B cell development is partially independent of its catalytic activity

NARCIS (Netherlands)

S. Middendorp; G.M. Dingjan (Gemma); A. Maas (Alex); K. Dahlenborg; R.W. Hendriks (Rudi)

2003-01-01

textabstractThe Tec family member Bruton's tyrosine kinase (Btk) is a cytoplasmic protein tyrosine kinase that transduces signals from the pre-B and B cell receptor (BCR). Btk is involved in pre-B cell maturation by regulating IL-7 responsiveness, cell surface phenotype changes,

DMPD: Receptor tyrosine kinases and the regulation of macrophage activation. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 14726496 Receptor tyrosine kinases and the regulation of macrophage activation. Cor...osine kinases and the regulation of macrophage activation. PubmedID 14726496 Title Receptor tyrosine...rell PH, Morrison AC, Lutz MA. J Leukoc Biol. 2004 May;75(5):731-7. Epub 2004 Jan 14. (.png) (.svg) (.html) (.csml) Show Receptor tyr

Dimethyl sulfoxide-caused changes in pro- and anti-angiogenic factor levels could contribute to an anti-angiogenic response in HeLa cells.

Science.gov (United States)

Şimşek, Ece; Aydemir, Esra Arslan; İmir, Nilüfer; Koçak, Orhan; Kuruoğlu, Aykut; Fışkın, Kayahan

2015-10-01

Dimethyl sulfoxide (DMSO) is widely used in biological research as a general solvent. While it has been previously demonstrated that DMSO possesses a wide range of pharmacological effects, there is no published work regarding the effects of DMSO on pro-angiogenic factor levels. This study was designed to investigate the possible effects of DMSO on the levels of three pro-angiogenic factors released from HeLa cells in vitro. Cells were treated with two different and previously determined concentrations of DMSO. The cytotoxic effects of DMSO concentrations on HeLa cells were determined via MTT. Survival rates of DMSO-treated cells were determined by Invitrogen live/dead viability/cytotoxicity kit and trypan blue exclusion assay. Changes in the pro-angiogenic levels in media were evaluated by Cayman's Substance P Enzyme Immunoassay ELISA kit. Vascular endothelial growth factor ELISA kit and interferon gamma ELISA kit for substance P, VEGF and IFNγ respectively. Changes in substance P levels were corrected by standard western blotting. Changes in VEGF and IFNγ levels were corrected both by western blot and real time PCR. Treatment with 1.4 μM DMSO caused a time-dependent inhibition of cell proliferation at 24, 48 and 72 h. 1.4 μM DMSO caused a significant reduction in VEGF levels at 72 h of incubation and sharp increases in IFNγ levels at both 48 and 72 h of incubation. According to real time PCR analyses, DMSO (1.4 μM) exhibited an inhibitory effect on VEGF but acted as an augmenter of IFNγ release on HeLa cells in vitro. This is the first report showing that the general solvent DMSO suppressed HeLa cell proliferation, decreased the levels of two pro-angiogenic factors (substance P and VEGF) and increased the release of an anti-angiogenic factor IFNγ in vitro. Copyright © 2015 Elsevier Ltd. All rights reserved.

Band 3 tyrosine kinase in avian erythrocyte plasma membrane is immunologically related to pp60c-src

International Nuclear Information System (INIS)

Hillsgrove, D.; Shores, C.G.; Parker, J.C.; Maness, P.F.

1987-01-01

The authors have identified in the plasma membrane of the chicken erythrocyte a 60-kDa tyrosine-specific protein kinase immunologically related to the transforming protein pp60 v-src of Rous sarcoma virus. The erythrocyte protein kinase phosphorylated heavy chains of tumor-bearing rabbit (TBR) antibodies reactive with pp60 c-src at tyrosine in immune complex protein kinase assays. The kinase was identified as a 60-kDa protein by [ 35 S]methionine labeling of erythrocytes and by autophosphorylation in immune complexes. The kinase migrated on two-dimensional gel electrophoresis with an apparent pI and molecular mass similar to pp60 c-src . A plasma membrane-enriched fraction isolated from chicken red cells contained the majority of the kinase activity. Incubation of the plasma membrane fraction with [ 32 P]ATP resulted in tyrosine phosphorylation of the anion transport protein band 3. Band 3 phosphorylation was blocked by TBR antibodies, indicting that the kinase recognized by pp60 c-src antibodies was responsible for band 3 phosphorylation. These results demonstrate that the avian erythrocyte plasma membrane contains a tightly bound tyrosine-specific protein kinase identical or closely related to pp60 c-src and that this kinase is responsible for band 3 phosphorylation in vitro

Free radical-mediated stimulation of tyrosine-specific protein kinase in rat liver plasma membrane

International Nuclear Information System (INIS)

Chan, T.M.; Tatoyan, A.; Cheng, E.; Shargill, N.S.; Pleta, M.

1986-01-01

Incorporation of 32 P from (γ- 32 P)-ATP into endogenous proteins of plasma membranes isolated from rat liver was significantly increased by several naphthoquinones including menadione. This apparent stimulation of membrane-associated protein kinase activity by these compounds was most striking (up to 6-7 fold) when the synthetic copolymers containing glutamate and tyrosine residues (4:1) was used as substrate. Since tyrosine residues are the only possible phosphate acceptor in the copolymers, the quinone-stimulated liver membrane protein kinase is most likely tyrosine specific. Although not required for protein kinase activity, dithiothreitol (DTT) was necessary for its stimulation by these quinonoid compounds. Hydrolysis of ATP was not significantly affected by quinones under the experimental conditions. Both menadione and vitamin k 5 increased phosphorylation of plasma membrane proteins of molecular weight 45 and 60 kd. The stimulatory effect of menadione on protein phosphorylation was prevented by the addition of superoxide dismutase. Dihydroxyfumerate, which spontaneously produces various radical species, and H 2 O 2 , also stimulated tyrosine-specific protein phosphorylation. DTT was also required for their full effect. It, therefore, appears that quinonone stimulation of tyrosine-specific protein phosphorylation is mediated by oxygen radicals

Resistance to cytotoxic and anti-angiogenic anticancer agents: similarities and differences.

NARCIS (Netherlands)

Broxterman, H.J.; Lankelma, J.; Hoekman, K.

2003-01-01

Intrinsic resistance to anticancer drugs, or resistance developed during chemotherapy, remains a major obstacle to successful treatment. This is the case both for resistance to cytotoxic agents, directed at malignant cells, and for resistance to anti-angiogenic agents, directed at non-malignant

Pharmacogenetics of telatinib, a VEGFR-2 and VEGFR-3 tyrosine kinase inhibitor, used in patients with solid tumors

NARCIS (Netherlands)

N. Steeghs (Neeltje); A.J. Gelderblom (Hans); J.A.M. Wessels (Judith); F.A.L.M. Eskens (Ferry); N. de Bont (Natasja); J.W. Nortier (Johan); H.J. Guchelaar (Henk Jan)

2011-01-01

textabstractSummary: Purpose Telatinib is an orally active small-molecule tyrosine kinase inhibitor of kinase insert domain receptor (KDR; VEGFR-2) and fms-related tyrosine kinase 4 (FLT4; VEGFR-3). This study aims at the identification of relationships between single nucleotide polymorphisms (SNPs)

Characterizing the angiogenic activity of patients with single ventricle physiology and aortopulmonary collateral vessels.

Science.gov (United States)

Sandeep, Nefthi; Uchida, Yutaka; Ratnayaka, Kanishka; McCarter, Robert; Hanumanthaiah, Sridhar; Bangoura, Aminata; Zhao, Zhen; Oliver-Danna, Jacqueline; Leatherbury, Linda; Kanter, Joshua; Mukouyama, Yoh-Suke

2016-04-01

Patients with single ventricle congenital heart disease often form aortopulmonary collateral vessels via an unclear mechanism. To gain insights into the pathogenesis of aortopulmonary collateral vessels, we correlated angiogenic factor levels with in vitro activity and angiographic aortopulmonary collateral assessment and examined whether patients with single ventricle physiology have increased angiogenic factors that can stimulate endothelial cell sprouting in vitro. In patients with single ventricle physiology (n = 27) and biventricular acyanotic control patients (n = 21), hypoxia-inducible angiogenic factor levels were measured in femoral venous and arterial plasma at cardiac catheterization. To assess plasma angiogenic activity, we used a 3-dimensional in vitro cell sprouting assay that recapitulates angiogenic sprouting. Aortopulmonary collateral angiograms were graded using a 4-point scale. Compared with controls, patients with single ventricle physiology had increased vascular endothelial growth factor (artery: 58.7 ± 1.2 pg/mL vs 35.3 ± 1.1 pg/mL, P collateral severity. We are the first to correlate plasma angiogenic factor levels with angiography and in vitro angiogenic activity in patients with single ventricle disease with aortopulmonary collaterals. Patients with single ventricle disease have increased stromal-derived factor 1-alpha and soluble fms-like tyrosine kinase-1, and their roles in aortopulmonary collateral formation require further investigation. Plasma factors and angiogenic activity correlate poorly with aortopulmonary collateral severity in patients with single ventricles, suggesting complex mechanisms of angiogenesis. Published by Elsevier Inc.

Modeling tumor-associated edema in gliomas during anti-angiogenic therapy and its impact on imageable tumor

Directory of Open Access Journals (Sweden)

Andrea eHawkins-Daarud

2013-04-01

Full Text Available Glioblastoma, the most aggressive form of primary brain tumor is predominantly assessed with gadolinium-enhanced T1-weighted (T1Gd and T2-weighted magnetic resonance imaging (MRI. Pixel intensity enhancement on the T1Gd image is understood to correspond to the gadolinium contrast agent leaking from the tumor-induced neovasculature, while hyperintensity on the T2/FLAIR images corresponds with edema and infiltrated tumor cells. None of these modalities directly show tumor cells; rather, they capture abnormalities in the microenvironment caused by the presence of tumor cells. Thus, assessing disease response after treatments impacting the microenvironment remains challenging through the obscuring lens of MR imaging. Anti-angiogenic therapies have been used in the treatment of gliomas with spurious results ranging from no apparent response to significant imaging improvement with the potential for extremely diffuse patterns of tumor recurrence on imaging and autopsy. Anti-angiogenic treatment normalizes the vasculature, effectively decreasing vessel permeability and thus reducing tumor-induced edema, drastically altering T2-weighted MRI. We extend a previously developed mathematical model of glioma growth to explicitly incorporate edema formation allowing us to directly characterize and potentially predict the effects of anti-angiogenics on imageable tumor growth. A comparison of simulated glioma growth and imaging enhancement with and without bevacizumab supports the current understanding that anti-angiogenic treatment can serve as a surrogate for steroids and the clinically-driven hypothesis that anti-angiogenic treatment may not have any significant effect on the growth dynamics of the overall tumor-cell populations. However, the simulations do illustrate a potentially large impact on the level of edematous extracellular fluid, and thus on what would be imageable on T2/FLAIR MR for tumors with lower proliferation rates.

Bacillus subtilis strain deficient for the protein-tyrosine kinase PtkA exhibits impaired DNA replication

DEFF Research Database (Denmark)

Petranovic, Dina; Michelsen, Ole; Zahradka, K

2007-01-01

Bacillus subtilis has recently come into the focus of research on bacterial protein-tyrosine phosphorylation, with several proteins kinases, phosphatases and their substrates identified in this Gram-positive model organism. B. subtilis protein-tyrosine phosphorylation system Ptk...... microscopy. B. subtilis cells lacking the kinase PtkA accumulated extra chromosome equivalents, exhibited aberrant initiation mass for DNA replication and an unusually long D period....

Src-family-tyrosine kinase Lyn is critical for TLR2-mediated NF-κB activation through the PI 3-kinase signaling pathway.

Science.gov (United States)

Toubiana, Julie; Rossi, Anne-Lise; Belaidouni, Nadia; Grimaldi, David; Pene, Frederic; Chafey, Philippe; Comba, Béatrice; Camoin, Luc; Bismuth, Georges; Claessens, Yann-Erick; Mira, Jean-Paul; Chiche, Jean-Daniel

2015-10-01

TLR2 has a prominent role in host defense against a wide variety of pathogens. Stimulation of TLR2 triggers MyD88-dependent signaling to induce NF-κB translocation, and activates a Rac1-PI 3-kinase dependent pathway that leads to transactivation of NF-κB through phosphorylation of the P65 NF-κB subunit. This transactivation pathway involves tyrosine phosphorylations. The role of the tyrosine kinases in TLR signaling is controversial, with discrepancies between studies using only chemical inhibitors and knockout mice. Here, we show the involvement of the tyrosine-kinase Lyn in TLR2-dependent activation of NF-κB in human cellular models, by using complementary inhibition strategies. Stimulation of TLR2 induces the formation of an activation cluster involving TLR2, CD14, PI 3-kinase and Lyn, and leads to the activation of AKT. Lyn-dependent phosphorylation of the p110 catalytic subunit of PI 3-kinase is essential to the control of PI 3-kinase biological activity upstream of AKT and thereby to the transactivation of NF-κB. Thus, Lyn kinase activity is crucial in TLR2-mediated activation of the innate immune response in human mononuclear cells. © The Author(s) 2015.

Variability in bioavailability of small molecular tyrosine kinase inhibitors

NARCIS (Netherlands)

Herbrink, Maikel; Nuijen, Bastiaan; Schellens, Jan H M; Beijnen, Jos H.

2015-01-01

Small molecular tyrosine kinase inhibitors (smTKIs) are in the centre of the very quickly expanding area of personalized chemotherapy and oral applicability thereof. The number of drugs in this class is rapidly growing, with twenty current approvals by both the European Medicines Agency (EMA) and

HER2-targeted therapy in breast cancer. Monoclonal antibodies and tyrosine kinase inhibitors

DEFF Research Database (Denmark)

Nielsen, Dorte Lisbet; Andersson, Michael; Kamby, Claus

2008-01-01

There is strong clinical evidence that trastuzumab, a monoclonal antibody targeting the human epidermal growth factor receptor (HER) two tyrosine kinase receptor, is an important component of first-line treatment of patients with HER2-positive metastatic breast cancer. In particular the combination...... of trastuzumab to chemotherapy improves disease-free and overall survival. The use of lapatinib, a dual tyrosine kinase inhibitor of both HER1 and HER2, in combination with capecitabine in the second-line treatment of HER2-positive patients with metastatic breast cancer previously treated with trastuzumab has...

Oligonucleotide aptamers against tyrosine kinase receptors: Prospect for anticancer applications.

Science.gov (United States)

Camorani, Simona; Crescenzi, Elvira; Fedele, Monica; Cerchia, Laura

2018-04-01

Transmembrane receptor tyrosine kinases (RTKs) play crucial roles in cancer cell proliferation, survival, migration and differentiation. Area of intense research is searching for effective anticancer therapies targeting these receptors and, to date, several monoclonal antibodies and small-molecule tyrosine kinase inhibitors have entered the clinic. However, some of these drugs show limited efficacy and give rise to acquired resistance. Emerging highly selective compounds for anticancer therapy are oligonucleotide aptamers that interact with their targets by recognizing a specific three-dimensional structure. Because of their nucleic acid nature, the rational design of advanced strategies to manipulate aptamers for both diagnostic and therapeutic applications is greatly simplified over antibodies. In this manuscript, we will provide a comprehensive overview of oligonucleotide aptamers as next generation strategies to efficiently target RTKs in human cancers. Copyright © 2018 Elsevier B.V. All rights reserved.

Expression, purification and preliminary crystallographic studies on the catalytic region of the nonreceptor tyrosine kinase Fes

International Nuclear Information System (INIS)

Gnemmi, Ilaria; Scotti, Claudia; Cappelletti, Donata; Canonico, Pier Luigi; Condorelli, Fabrizio; Rosano, Camillo

2006-01-01

The catalytic domain of human Fes tyrosine kinase has been cloned, expressed, purified and crystallized. The proto-oncogene tyrosine protein kinase c-fps/fes encodes a structurally unique protein (Fes) of the nonreceptor protein-tyrosine kinase (PTK) family. Its expression has been demonstrated in myeloid haematopoietic cells, vascular endothelial cells and in neurons. In human-derived and murine-derived cell lines, the activated form of this kinase can induce cellular transformation; moreover, it has been shown that Fes is involved in the regulation of cell–cell and cell–matrix interactions mediated by adherens junctions and focal adhesions. The N-terminus of Fes contains the FCH (Fps/Fes/Fer/CIP4 homology) domain, which is unique to the Fes/Fer kinase family. It is followed by three coiled-coil domains and an SH2 (Src-homology 2) domain. The catalytic region (Fes-CR) is located at the C-terminus of the protein. The successful expression, purification and crystallization of the catalytic part of Fes (Fes-CR) are described

Targeted Proteomics to Assess the Response to Anti-Angiogenic Treatment in Human Glioblastoma (GBM).

Science.gov (United States)

Demeure, Kevin; Fack, Fred; Duriez, Elodie; Tiemann, Katja; Bernard, Amandine; Golebiewska, Anna; Bougnaud, Sébastien; Bjerkvig, Rolf; Domon, Bruno; Niclou, Simone P

2016-02-01

Glioblastoma (GBM) is a highly aggressive primary brain tumor with dismal outcome for affected patients. Because of the significant neo-angiogenesis exhibited by GBMs, anti-angiogenic therapies have been intensively evaluated during the past years. Recent clinical studies were however disappointing, although a subpopulation of patients may benefit from such treatment. We have previously shown that anti-angiogenic targeting in GBM increases hypoxia and leads to a metabolic adaptation toward glycolysis, suggesting that combination treatments also targeting the glycolytic phenotype may be effective in GBM patients. The aim of this study was to identify marker proteins that are altered by treatment and may serve as a short term readout of anti-angiogenic therapy. Ultimately such proteins could be tested as markers of efficacy able to identify patient subpopulations responsive to the treatment. We applied a proteomics approach based on selected reaction monitoring (SRM) to precisely quantify targeted protein candidates, selected from pathways related to metabolism, apoptosis and angiogenesis. The workflow was developed in the context of patient-derived intracranial GBM xenografts developed in rodents and ensured the specific identification of human tumor versus rodent stroma-derived proteins. Quality control experiments were applied to assess sample heterogeneity and reproducibility of SRM assays at different levels. The data demonstrate that tumor specific proteins can be precisely quantified within complex biological samples, reliably identifying small concentration differences induced by the treatment. In line with previous work, we identified decreased levels of TCA cycle enzymes, including isocitrate dehydrogenase, whereas malectin, calnexin, and lactate dehydrogenase A were augmented after treatment. We propose the most responsive proteins of our subset as potential novel biomarkers to assess treatment response after anti-angiogenic therapy that warrant future

Expression of tyrosine kinase gene in mouse thymic stromal cells

NARCIS (Netherlands)

Rinke de Wit, T. F.; Izon, D. J.; Revilla, C.; Oosterwegel, M.; Bakker, A. Q.; van Ewijk, W.; Kruisbeek, A. M.

1996-01-01

Amongst the most important signal transduction molecules involved in regulating growth and differentiation are the protein tyrosine kinases (PTK). Since T cell development is a consequence of interactions between thymic stromal cells (TSC) and thymocytes, identification of the PTK in both

Signaling by Kit protein-tyrosine kinase--the stem cell factor receptor.

Science.gov (United States)

Roskoski, Robert

2005-11-11

Signaling by stem cell factor and Kit, its receptor, plays important roles in gametogenesis, hematopoiesis, mast cell development and function, and melanogenesis. Moreover, human and mouse embryonic stem cells express Kit transcripts. Stem cell factor exists as both a soluble and a membrane-bound glycoprotein while Kit is a receptor protein-tyrosine kinase. The complete absence of stem cell factor or Kit is lethal. Deficiencies of either produce defects in red and white blood cell production, hypopigmentation, and sterility. Gain-of-function mutations of Kit are associated with several human neoplasms including acute myelogenous leukemia, gastrointestinal stromal tumors, and mastocytomas. Kit consists of an extracellular domain, a transmembrane segment, a juxtamembrane segment, and a protein kinase domain that contains an insert of about 80 amino acid residues. Binding of stem cell factor to Kit results in receptor dimerization and activation of protein kinase activity. The activated receptor becomes autophosphorylated at tyrosine residues that serve as docking sites for signal transduction molecules containing SH2 domains. The adaptor protein APS, Src family kinases, and Shp2 tyrosyl phosphatase bind to phosphotyrosine 568. Shp1 tyrosyl phosphatase and the adaptor protein Shc bind to phosphotyrosine 570. C-terminal Src kinase homologous kinase and the adaptor Shc bind to both phosphotyrosines 568 and 570. These residues occur in the juxtamembrane segment of Kit. Three residues in the kinase insert domain are phosphorylated and attract the adaptor protein Grb2 (Tyr703), phosphatidylinositol 3-kinase (Tyr721), and phospholipase Cgamma (Tyr730). Phosphotyrosine 900 in the distal kinase domain binds phosphatidylinositol 3-kinase which in turn binds the adaptor protein Crk. Phosphotyrosine 936, also in the distal kinase domain, binds the adaptor proteins APS, Grb2, and Grb7. Kit has the potential to participate in multiple signal transduction pathways as a result of

Tyrosine kinase, aurora kinase and leucine aminopeptidase as attractive drug targets in anticancer therapy - characterisation of their inhibitors.

Science.gov (United States)

Ziemska, Joanna; Solecka, Jolanta

Cancers are the leading cause of deaths all over the world. Available anticancer agents used in clinics exhibit low therapeutic index and usually high toxicity. Wide spreading drug resistance of cancer cells induce a demanding need to search for new drug targets. Currently, many on-going studies on novel compounds with potent anticancer activity, high selectivity as well as new modes of action are conducted. In this work, we describe in details three enzyme groups, which are at present of extensive interest to medical researchers and pharmaceutical companies. These include receptor tyrosine kinases (e.g. EGFR enzymes) and non-receptor tyrosine kinases (Src enzymes), type A, B and C Aurora kinases and aminopeptidases, especially leucine aminopeptidase. We discuss classification of these enzymes, biochemistry as well as their role in the cell cycle under normal conditions and during cancerogenesis. Further on, the work describes enzyme inhibitors that are under in vitro, preclinical, clinical studies as well as drugs available on the market. Both, chemical structures of discovered inhibitors and the role of chemical moieties in novel drug design are discussed. Described enzymes play essential role in cell cycle, especially in mitosis (Aurora kinases), cell differentiation, growth and apoptosis (tyrosine kinases) as well as G1/S transition (leucine aminopeptidase). In cancer cells, they are overexpressed and only their inhibition may stop tumor progression. This review presents the clinical outcomes of selected inhibitors and argues the safety of drug usage in human volunteers. Clinical studies of EGFR and Src kinase inhibitors in different tumors clearly show the need for molecular selection of patients (to those with mutations in genes coding EGFR and Src) to achieve positive clinical response. Current data indicates the great necessity for new anticancer treatment and actions to limit off-target activity.

Anti-angiogenic SPARC peptides inhibit progression of neuroblastoma tumors

Directory of Open Access Journals (Sweden)

Tian Yufeng

2010-06-01

Full Text Available Abstract Background New, more effective strategies are needed to treat highly aggressive neuroblastoma. Our laboratory has previously shown that full-length Secreted Protein Acidic and Rich in Cysteine (SPARC and a SPARC peptide corresponding to the follistatin domain of the protein (FS-E potently block angiogenesis and inhibit the growth of neuroblastoma tumors in preclinical models. Peptide FS-E is structurally complex and difficult to produce, limiting its potential as a therapeutic in the clinic. Results In this study, we synthesized two smaller and structurally more simple SPARC peptides, FSEN and FSEC, that respectively correspond to the N-and C-terminal loops of peptide FS-E. We show that both peptides FSEN and FSEC have anti-angiogenic activity in vitro and in vivo, although FSEC is more potent. Peptide FSEC also significantly inhibited the growth of neuroblastoma xenografts. Histologic examination demonstrated characteristic features of tumor angiogenesis with structurally abnormal, tortuous blood vessels in control neuroblastoma xenografts. In contrast, the blood vessels observed in tumors, treated with SPARC peptides, were thin walled and structurally more normal. Using a novel method to quantitatively assess blood vessel abnormality we demonstrated that both SPARC peptides induced changes in blood vessel architecture that are consistent with blood vessel normalization. Conclusion Our results demonstrate that SPARC peptide FSEC has potent anti-angiogenic and anti-tumorigenic effects in neuroblastoma. Its simple structure and ease of production indicate that it may have clinical utility in the treatment of high-risk neuroblastoma and other types of pediatric and adult cancers, which depend on angiogenesis.

Engineering of kinase-based protein interacting devices: active expression of tyrosine kinase domains

KAUST Repository

Diaz Galicia, Miriam Escarlet

2018-05-01

Protein-protein interactions modulate cellular processes in health and disease. However, tracing weak or rare associations or dissociations of proteins is not a trivial task. Kinases are often regulated through interaction partners and, at the same time, themselves regulate cellular interaction networks. The use of kinase domains for creating a synthetic sensor device that reads low concentration protein-protein interactions and amplifies them to a higher concentration interaction which is then translated into a FRET (Fluorescence Resonance Energy Transfer) signal is here proposed. To this end, DNA constructs for interaction amplification (split kinases), positive controls (intact kinase domains), scaffolding proteins and phosphopeptide - SH2-domain modules for the reading of kinase activity were assembled and expression protocols for fusion proteins containing Lyn, Src, and Fak kinase domains in bacterial and in cell-free systems were optimized. Also, two non-overlapping methods for measuring the kinase activity of these proteins were stablished and, finally, a protein-fragment complementation assay with the split-kinase constructs was tested. In conclusion, it has been demonstrated that features such as codon optimization, vector design and expression conditions have an impact on the expression yield and activity of kinase-based proteins. Furthermore, it has been found that the defined PURE cell-free system is insufficient for the active expression of catalytic kinase domains. In contrast, the bacterial co-expression with phosphatases produced active kinase fusion proteins for two out of the three tested Tyrosine kinase domains.

Phosphopeptide occupancy and photoaffinity cross-linking of the v-Src SH2 domain attenuates tyrosine kinase activity.

Science.gov (United States)

Garcia, P; Shoelson, S E; Drew, J S; Miller, W T

1994-12-02

Phosphorylation of c-Src at carboxyl-terminal Tyr-527 suppresses tyrosine kinase activity and transforming potential, presumably by facilitating the intramolecular interaction of the C terminus of Src with its SH2 domain. In addition, it has been shown previously that occupancy of the c-Src SH2 domain with a phosphopeptide stimulates c-Src kinase catalytic activity. We have performed analogous studies with v-Src, the transforming protein from Rous sarcoma virus, which has extensive homology with c-Src. v-Src lacks an autoregulatory phosphorylation site, and its kinase domain is constitutively active. Phosphopeptides corresponding to the sequences surrounding c-Src Tyr-527 and a Tyr-Glu-Glu-Ile motif from the hamster polyoma virus middle T antigen inhibit tyrosine kinase activity of baculovirus-expressed v-Src 2- and 4-fold, respectively. To determine the mechanism of this regulation, the Tyr-527 phosphopeptide was substituted with the photoactive amino acid p-benzoylphenylalanine at the adjacent positions (N- and C-terminal) to phosphotyrosine. These peptides photoinactivate the v-Src tyrosine kinase 5-fold in a time- and concentration-dependent manner. Furthermore, the peptides cross-link an isolated Src SH2 domain with similar rates and specificity. These data indicate that occupancy of the v-Src SH2 domain induces a conformational change that is transmitted to the kinase domain and attenuates tyrosine kinase activity.

Anti-migratory effect of vinflunine in endothelial and glioblastoma cells is associated with changes in EB1 C-terminal detyrosinated/tyrosinated status.

Directory of Open Access Journals (Sweden)

Amandine Rovini

Full Text Available We previously showed that vinflunine, a microtubule-targeting drug of the Vinca-alkaloid family exerted its anti-angiogenic/anti-migratory activities through an increase in microtubule dynamics and an inhibition of microtubule targeting to adhesion sites. Such effect was associated with a reduction of EB1 comet length at microtubule (+ ends. In this work we first showed that the pro-angiogenic vascular endothelial growth factor VEGF suppressed microtubule dynamics in living Human Umbilical Vein Endothelial Cells (HUVECs, increased EB1 comet length by 40%, and induced EB1 to bind all along the microtubules, without modifying its expression level. Such microtubule (+ end stabilization occurred close to the plasma membrane in the vicinity of focal adhesion as shown by TIRF microscopy experiments. Vinflunine completely abolished the effect of VEGF on EB1 comets. Interestingly, we found a correlation between the reduction of EB1 comet length by vinflunine and the inhibition of cell migration. By using 2D gel electrophoresis we demonstrated for the first time that EB1 underwent several post-translational modifications in endothelial and tumor cells. Particularly, the C-terminal EEY sequence was poorly detectable in control and VEGF-treated HUVECs suggesting the existence of a non-tyrosinated form of EB1. By using specific antibodies that specifically recognized and discriminated the native tyrosinated form of EB1 and a putative C-terminal detyrosinated form, we showed that a detyrosinated form of EB1 exists in HUVECs and tumor cells. Interestingly, vinflunine decreased the level of the detyrosinated form and increased the native tyrosinated form of EB1. Using 3-L-Nitrotyrosine incorporation experiments, we concluded that the EB1 C-terminal modifications result from a detyrosination/retyrosination cycle as described for tubulin. Altogether, our results show that vinflunine inhibits endothelial cell migration through an alteration of EB1 comet length

Inhibitors of dual-specificity tyrosine phosphorylation-regulated kinases (DYRK) exert a strong anti-herpesviral activity.

Science.gov (United States)

Hutterer, Corina; Milbradt, Jens; Hamilton, Stuart; Zaja, Mirko; Leban, Johann; Henry, Christophe; Vitt, Daniel; Steingruber, Mirjam; Sonntag, Eric; Zeitträger, Isabel; Bahsi, Hanife; Stamminger, Thomas; Rawlinson, William; Strobl, Stefan; Marschall, Manfred

2017-07-01

Infection with human cytomegalovirus (HCMV) is a serious medical problem, particularly in immunocompromised individuals and neonates. The success of (val)ganciclovir therapy is hampered by low drug compatibility and induction of viral resistance. A novel strategy of antiviral treatment is based on the exploitation of cell-directed signaling, e. g. pathways with a known relevance for carcinogenesis and tumor drug development. Here we describe a principle for putative antiviral drugs based on targeting dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs). DYRKs constitute an evolutionarily conserved family of protein kinases with key roles in the control of cell proliferation and differentiation. Members of the DYRK family are capable of phosphorylating a number of substrate proteins, including regulators of the cell cycle, e.g. DYRK1B can induce cell cycle arrest, a critical step for the regulation of HCMV replication. Here we provide first evidence for a critical role of DYRKs during viral replication and the high antiviral potential of DYRK inhibitors (SC84227, SC97202 and SC97208, Harmine and AZ-191). Using established replication assays for laboratory and clinically relevant strains of HCMV, concentration-dependent profiles of inhibition were obtained. Mean inhibitory concentrations (EC50) of 0.98 ± 0.08 μM/SC84227, 0.60 ± 0.02 μM/SC97202, 6.26 ± 1.64 μM/SC97208, 0.71 ± 0.019 μM/Harmine and 0.63 ± 0.23 μM/AZ-191 were determined with HCMV strain AD169-GFP for the infection of primary human fibroblasts. A first analysis of the mode of antiviral action suggested a block of viral replication at the early-late stage of HCMV gene expression. Moreover, rhesus macaque cytomegalovirus (RhCMV), varicella-zoster virus (VZV) and herpes simplex virus (HSV-1) showed a similarly high sensitivity to these compounds. Thus, we conclude that DYRK signaling represents a promising target pathway for the development of novel anti

Significant blockade of multiple receptor tyrosine kinases by MGCD516 (Sitravatinib), a novel small molecule inhibitor, shows potent anti-tumor activity in preclinical models of sarcoma.

Science.gov (United States)

Patwardhan, Parag P; Ivy, Kathryn S; Musi, Elgilda; de Stanchina, Elisa; Schwartz, Gary K

2016-01-26

Sarcomas are rare but highly aggressive mesenchymal tumors with a median survival of 10-18 months for metastatic disease. Mutation and/or overexpression of many receptor tyrosine kinases (RTKs) including c-Met, PDGFR, c-Kit and IGF1-R drive defective signaling pathways in sarcomas. MGCD516 (Sitravatinib) is a novel small molecule inhibitor targeting multiple RTKs involved in driving sarcoma cell growth. In the present study, we evaluated the efficacy of MGCD516 both in vitro and in mouse xenograft models in vivo. MGCD516 treatment resulted in significant blockade of phosphorylation of potential driver RTKs and induced potent anti-proliferative effects in vitro. Furthermore, MGCD516 treatment of tumor xenografts in vivo resulted in significant suppression of tumor growth. Efficacy of MGCD516 was superior to imatinib and crizotinib, two other well-studied multi-kinase inhibitors with overlapping target specificities, both in vitro and in vivo. This is the first report describing MGCD516 as a potent multi-kinase inhibitor in different models of sarcoma, superior to imatinib and crizotinib. Results from this study showing blockade of multiple driver signaling pathways provides a rationale for further clinical development of MGCD516 for the treatment of patients with soft-tissue sarcoma.

Interaction between focal adhesion kinase and Crk-associated tyrosine kinase substrate p130Cas.

Science.gov (United States)

Polte, T R; Hanks, S K

1995-11-07

The focal adhesion kinase (FAK) has been implicated in integrin-mediated signaling events and in the mechanism of cell transformation by the v-Src and v-Crk oncoproteins. To gain further insight into FAK signaling pathways, we used a two-hybrid screen to identify proteins that interact with mouse FAK. The screen identified two proteins that interact with FAK via their Src homology 3 (SH3) domains: a v-Crk-associated tyrosine kinase substrate (Cas), p130Cas, and a still uncharacterized protein, FIPSH3-2, which contains an SH3 domain closely related to that of p130Cas. These SH3 domains bind to the same proline-rich region of FAK (APPKPSR) encompassing residues 711-717. The mouse p130Cas amino acid sequence was deduced from cDNA clones, revealing an overall high degree of similarity to the recently reported rat sequence. Coimmunoprecipitation experiments confirmed that p130Cas and FAK are associated in mouse fibroblasts. The stable interaction between p130Cas and FAK emerges as a likely key element in integrin-mediated signal transduction and further represents a direct molecular link between the v-Src and v-Crk oncoproteins. The Src family kinase Fyn, whose Src homology 2 (SH2) domain binds to the major FAK autophosphorylation site (tyrosine 397), was also identified in the two-hybrid screen.

Negative Regulation of Receptor Tyrosine Kinase (RTK Signaling: A Developing Field

Directory of Open Access Journals (Sweden)

Fernanda Ledda

2007-01-01

Full Text Available ophic factors control cellular physiology by activating specific receptor tyrosine kinases (RTKs. While the over activation of RTK signaling pathways is associated with cell growth and cancer, recent findings support the concept that impaired down-regulation or deactivation of RTKs may also be a mechanism involved in tumor formation. Under this perspective, the molecular determinants of RTK signaling inhibition may act as tumor-suppressor genes and have a potential role as tumor markers to monitor and predict disease progression. Here, we review the current understanding of the physiological mechanisms that attenuate RTK signaling and discuss evidence that implicates deregulation of these events in cancer.Abbreviations: BDP1: Brain-derived phosphatase 1; Cbl: Casitas B-lineage lymphoma; CIN-85: Cbl-interacting protein of 85 kDa; DER: Drosophila EGFR; EGFR: Epidermal growth factor receptor; ERK 1/2: Extracellular signal-regulated kinase 1/2; Grb2: Growth factor receptor-bound protein 2; HER2: Human epidermal growth factor receptor 2; LRIG: Leucine-rich repeats and immunoglobulin-like domain 1; MAPK: Mitogen-activated protein kinase; Mig 6: Mitogen-inducible gene 6; PTEN: Phosphatase and tensin homologue; RET: Rearranged in transformation; RTK: Receptor tyrosine kinase. SH2 domain: Src-homology 2 domain; SH3 domain: Src-homology 3 domain; Spry: Sprouty.

Tyrosine kinase inhibition: A therapeutic target for the management of chronic-phase chronic myeloid leukemia

Science.gov (United States)

Jabbour, Elias J; Cortes, Jorge E; Kantarjian, Hagop M

2014-01-01

Chronic myeloid leukemia (CML) is a hematologic neoplasm with a progressive, ultimately terminal, disease course. In most cases, CML arises owing to the aberrant formation of a chimeric gene for a constitutively active tyrosine kinase. Inhibition of the signaling activity of this kinase has proved to be a highly successful treatment target transforming the prognosis of patients with CML. New tyrosine kinase inhibitors (TKIs) continue to improve the management of CML, offering alternative options for those resistant to or intolerant of standard TKIs. Here we review the pathobiology of CML and explore emerging strategies to optimize the management of chronic-phase CML, particularly first-line treatment. PMID:24236822

Effects of tyrosine kinase and phosphatase inhibitors on mitosis progression in synchronized tobacco BY-2 cells.

Science.gov (United States)

Sheremet, Ya A; Yemets, A I; Azmi, A; Vissenberg, K; Verbelen, J P; Blume, Ya B

2012-01-01

To test whether reversible tubulin phosphorylation plays any role in the process of plant mitosis the effects of inhibitors of tyrosine kinases, herbimycin A, genistein and tyrphostin AG 18, and of an inhibitor of tyrosine phosphatases, sodium orthovanadate, on microtubule organization and mitosis progression in a synchronized BY-2 culture has been investigated. It was found that treatment with inhibitors of tyrosine kinases of BY-2 cells at the G2/M transition did not lead to visible disturbances of mitotic microtubule structures, while it did reduce the frequency of their appearance. We assume that a decreased tyrosine phosphorylation level could alter the microtubule dynamic instability parameters during interphase/prophase transition. All types of tyrosine kinase inhibitors used caused a prophase delay: herbimycin A and genistein for 2 h, and tyrphostin AG18 for 1 h. Thereafter the peak of mitosis was displaced for 1 h by herbimycin A or genistein exposure, but after tyrphostin AG18 treatment the timing of the mitosis-peak was comparable to that in control cells. Enhancement of tyrosine phosphorylation induced by the tyrosine phosphatase inhibitor resulted in the opposite effect on BY-2 mitosis transition. Culture treatment with sodium orthovanadate during 1 h resulted in an accelerated start of the prophase and did not lead to the alteration in time of the mitotic index peak formation, as compared to control cells. We suppose that the reversible tyrosine phosphorylation can be involved in the regulation of interphase to M phase transition possibly through regulation of microtubule dynamics in plant cells.

Suppressor of cytokine signaling 1 interacts with oncogenic lymphocyte-specific protein tyrosine kinase.

Science.gov (United States)

Venkitachalam, Srividya; Chueh, Fu-Yu; Leong, King-Fu; Pabich, Samantha; Yu, Chao-Lan

2011-03-01

Lymphocyte-specific protein tyrosine kinase (Lck) plays a key role in T cell signal transduction and is tightly regulated by phosphorylation and dephosphorylation. Lck can function as an oncoprotein when overexpressed or constantly activated by mutations. Our previous studies showed that Lck-induced cellular transformation could be suppressed by enforced expression of suppressor of cytokine signaling 1 (SOCS1), a SOCS family member involved in the negative feedback control of cytokine signaling. We observed attenuated Lck kinase activity in SOCS1-expressing cells, suggesting an important role of SOCS in regulating Lck functions. It remains largely unknown whether and how SOCS proteins interact with the oncogenic Lck kinase. Here, we report that among four SOCS family proteins, SOCS1, SOCS2, SOCS3 and CIS (cytokine-inducible SH2 domain containing protein), SOCS1 has the highest affinity in binding to the oncogenic Lck kinase. We identified the positive regulatory phosphotyrosine 394 residue in the kinase domain as the key interacting determinant in Lck. Additionally, the Lck kinase domain alone is sufficient to bind SOCS1. While the SH2 domain in SOCS1 is important in its association with the oncogenic Lck kinase, other functional domains may also contribute to overall binding affinity. These findings provide important mechanistic insights into the role of SOCS proteins as tumor suppressors in cells transformed by oncogenic protein tyrosine kinases.

Intramolecular regulatory switch in ZAP-70: analogy with receptor tyrosine kinases

Czech Academy of Sciences Publication Activity Database

Brdička, Tomáš; Kadlecek, T.A.; Roose, J.P.; Pastuszak, A.W.; Weiss, A.

2005-01-01

Roč. 25, č. 12 (2005), s. 4924-4933 ISSN 0270-7306 Institutional research plan: CEZ:AV0Z50520514 Keywords : protein tyrosin- kinase * phosphorylation * ZAP-70 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 7.093, year: 2005

Angiogenic competency of biodegradable hydrogels fabricated from polyethylene glycol-crosslinked tyrosine-derived polycarbonates

Directory of Open Access Journals (Sweden)

HJ Sung

2008-04-01

Full Text Available Synthetic biomaterials can be used as instructive biological milieus to guide cellular behaviour and function. To further realize this application, we synthesized a series of structurally similar hydrogels and tested their ability to modulate angiogenesis. Hydrogels were synthesized from poly(DTE-co-x% DT carbonate crosslinked by y% poly(ethylene glycol (PEG. Hydrogel desaminotyrosyl tyrosine (DT contents (x% ranged from 10-100%, and crosslink densities (y% PEG-crosslinker ranged from 5-80%. The hydrogels were fashioned into porous scaffolds with highly interconnected macro- and micro-pore (>100 and <10 mm in diameter, respectively architecture using poly(DTE-co-10%DT carbonate crosslinked with 8% PEG. Under physiological conditions (in vitro, the hydrogels degraded into three major products: desaminotyrosyl-tyrosine ethyl ester (DTE, desaminotyrosyl tyrosine (DT, and poly(ethylene glycol-di-DT-hydrazide (PEG-di-DT hydrazide. Increasing either DT content or crosslink density brought quickened degradation. Because DT and DTE, two of the three major degradation products, have not demonstrated any noticeable cytotoxicity or angiogenic effect in previous studies, we measured the cytotoxicity of PEG-di-DT hydrazide, the third major degradation product. We found that PEG-di-DT hydrazide only displayed significant cytotoxicity at the high concentration of 100 mg/mL. Interestingly, PEG-di-DT hydrazide and its further degradation product PEG-dihydrazide stimulated in vitro endothelial cell migration and tubulogenesis, which is comparable to results found with FGF-beta treatment. Subcutaneous implantation of the PEG-crosslinked poly(DTE-co-10%DT carbonate scaffolds into the backs of rats elicited greater tissue growth over time and superior vascularization than poly(DTE carbonate implantation. These results show that this new class of biomaterials has a strong potential to modulate angiogenesis.

Phorbol ester-induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity.

Science.gov (United States)

Takayama, S; White, M F; Kahn, C R

1988-03-05

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the function of the insulin receptor was examined in intact hepatoma cells (Fao) and in solubilized extracts purified by wheat germ agglutinin chromatography. Incubation of ortho[32P]phosphate-labeled Fao cells with TPA increased the phosphorylation of the insulin receptor 2-fold after 30 min. Analysis of tryptic phosphopeptides from the beta-subunit of the receptor by reverse-phase high performance liquid chromatography and determination of their phosphoamino acid composition suggested that TPA predominantly stimulated phosphorylation of serine residues in a single tryptic peptide. Incubation of the Fao cells with insulin (100 nM) for 1 min stimulated 4-fold the phosphorylation of the beta-subunit of the insulin receptor. Prior treatment of the cells with TPA inhibited the insulin-stimulated tyrosine phosphorylation by 50%. The receptors extracted with Triton X-100 from TPA-treated Fao cells and purified on immobilized wheat germ agglutinin retained the alteration in kinase activity and exhibited a 50% decrease in insulin-stimulated tyrosine autophosphorylation and phosphotransferase activity toward exogenous substrates. This was due primarily to a decrease in the Vmax for these reactions. TPA treatment also decreased the Km of the insulin receptor for ATP. Incubation of the insulin receptor purified from TPA-treated cells with alkaline phosphatase decreased the phosphate content of the beta-subunit to the control level and reversed the inhibition, suggesting that the serine phosphorylation of the beta-subunit was responsible for the decreased tyrosine kinase activity. Our results support the notion that the insulin receptor is a substrate for protein kinase C in the Fao cell and that the increase in serine phosphorylation of the beta-subunit of the receptor produced by TPA treatment inhibited tyrosine kinase activity in vivo and in vitro. These data suggest that protein kinase C may regulate the function

[Recent Advances and Prospect of Advanced Non-small Cell Lung Cancer Targeted 
Therapy: Focus on Small Molecular Tyrosine Kinase Inhibitors].

Science.gov (United States)

Zhang, Guowei; Wang, Huijuan; Ma, Zhiyong

2017-04-20

At present the treatment of advanced non-small cell lung cancer enters a targeted era and develops rapidly. New drugs appear constantly. Small molecular tyrosine kinase inhibitors have occupied the biggest piece of the territory, which commonly have a clear biomarker as predictor, and show remarkable effect in specific molecular classification of patients. The epidermal growth factor tyrosine kinase inhibitors such as gefitinib, erlotinib, icotinib and anaplastic lymphoma kinase tyrosine kinase inhibitors crizotinib have brought a milestone advance. In recent years new generations of tyrosine kinase inhibitors have achieved a great success in patients with acquired resistance to the above two kinds of drugs. At the same time new therapeutic targets are constantly emerging. So in this paper, we reviewed and summarized the important drugs and clinical trails on this topic, and made a prospect of the future development.

Recent Advances and Prospect of Advanced Non-small Cell Lung Cancer Targeted 
Therapy: Focus on Small Molecular Tyrosine Kinase Inhibitors

Directory of Open Access Journals (Sweden)

Guowei ZHANG

2017-04-01

Full Text Available At present the treatment of advanced non-small cell lung cancer enters a targeted era and develops rapidly. New drugs appear constantly. Small molecular tyrosine kinase inhibitors have occupied the biggest piece of the territory, which commonly have a clear biomarker as predictor, and show remarkable effect in specific molecular classification of patients. The epidermal growth factor tyrosine kinase inhibitors such as gefitinib, erlotinib, icotinib and anaplastic lymphoma kinase tyrosine kinase inhibitors crizotinib have brought a milestone advance. In recent years new generations of tyrosine kinase inhibitors have achieved a great success in patients with acquired resistance to the above two kinds of drugs. At the same time new therapeutic targets are constantly emerging. So in this paper, we reviewed and summarized the important drugs and clinical trails on this topic, and made a prospect of the future development.

Assessment of the anti-angiogenic, anti-inflammatory and antinociceptive properties of ethyl vanillin.

Science.gov (United States)

Jung, Hyun-Joo; Song, Yun Seon; Kim, Kyunghoon; Lim, Chang-Jin; Park, Eun-Hee

2010-02-01

The present work aimed to assess novel pharmacological properties of ethyl vanillin (EVA) which is used as a flavoring agent for cakes, dessert, confectionary, etc. EVA exhibited an inhibitory activity in the chorioallantoic membrane angiogenesis. Anti-inflammatory activity of EVA was convinced using the two in vivo models, such as vascular permeability and air pouch models in mice. Antinociceptive activity of EVA was assessed using acetic acid-induced writhing model in mice. EVA suppressed production of nitric oxide and induction of inducible nitric oxide synthase in the lipopolysaccharide (LPS)-activated RAW264.7 macrophage cells. However, EVA could not suppress induction of cyclooxygenase-2 in the LPS-activated macrophages. EVA diminished reactive oxygen species level in the LPS-activated macrophages. EVA also suppressed enhanced matrix metalloproteinase-9 gelatinolytic activity in the LPSactivated RAW264.7 macrophage cells. EVA at the used concentrations couldn't diminish viability of the macrophage cells. Taken together, the anti-angiogenic, anti-inflammatory and anti-nociceptive properties of EVA are based on its suppressive effect on the production of nitric oxide possibly via decreasing the reactive oxygen species level.

Axl receptor tyrosine kinase is up-regulated in metformin resistant prostate cancer cells

Science.gov (United States)

Bansal, Nitu; Mishra, Prasun J.; Stein, Mark; DiPaola, Robert S.; Bertino, Joseph R.

2015-01-01

Recent epidemiological studies showed that metformin, a widely used anti-diabetic drug might prevent certain cancers. Metformin also has an anti-proliferative effect in preclinical studies of both hematologic malignancies as well as solid cancers and clinical studies testing metformin as an anti-cancer drug are in progress. However, all cancer types do not respond to metformin with the same effectiveness or acquire resistance. To understand the mechanism of acquired resistance and possibly its mechanism of action as an anti-proliferative agent, we developed metformin resistant LNCaP prostate cancer cells. Metformin resistant LNCaP cells had an increased proliferation rate, increased migration and invasion ability as compared to the parental cells, and expressed markers of epithelial-mesenchymal transition (EMT). A detailed gene expression microarray comparing the resistant cells to the wild type cells revealed that Edil2, Ereg, Axl, Anax2, CD44 and Anax3 were the top up-regulated genes and calbindin 2 and TPTE (transmembrane phosphatase with tensin homology) and IGF1R were down regulated. We focused on Axl, a receptor tyrosine kinase that has been shown to be up regulated in several drug resistance cancers. Here, we show that the metformin resistant cell line as well as castrate resistant cell lines that over express Axl were more resistant to metformin, as well as to taxotere compared to androgen sensitive LNCaP and CWR22 cells that do not overexpress Axl. Forced overexpression of Axl in LNCaP cells decreased metformin and taxotere sensitivity and knockdown of Axl in resistant cells increased sensitivity to these drugs. Inhibition of Axl activity by R428, a small molecule Axl kinase inhibitor, sensitized metformin resistant cells that overexpressed Axl to metformin. Inhibitors of Axl may enhance tumor responses to metformin and other chemotherapy in cancers that over express Axl. PMID:26036314

In vitro and in vivo anti-angiogenic activity of girinimbine isolated from Murraya koenigii

Directory of Open Access Journals (Sweden)

Iman V

2015-03-01

Full Text Available Venoos Iman,1 Hamed Karimian,1 Syam Mohan,2 Yahya Hasan Hobani,2 Mohamed Ibrahim Noordin,1 Mohd Rais Mustafa,3 Suzita Mohd Noor41Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia; 2Medical Research Center, University of Jazan, Jazan, Saudi Arabia; 3Department of Pharmacology, Centre for Natural Products and Drug Discovery (CENAR, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia; 4Department of Biomedical Science, Faculty of Medicine, University of Malaya, Kuala Lumpur, MalaysiaAbstract: Girinimbine is a carbazole alkaloid isolated from the stem bark and root of Murraya koenigii. Here we report that girinimbine is an inhibitor of angiogenic activity both in vitro and in vivo. MTT results showed that girinimbine inhibited proliferation of human umbilical vein endothelial cells, while results from endothelial cell invasion, migration, tube formation, and wound healing assays demonstrated significant time- and dose-dependent inhibition by girinimbine. A proteome profiler array done on girinimbine-treated human umbilical vein endothelial cells showed that girinimbine had mediated regulation of pro-angiogenic and anti-angiogenic proteins. The anti-angiogenic potential of girinimbine was also evidenced in vivo in the zebrafish embryo model wherein girinimbine inhibited neo vessel formation in zebrafish embryos following 24 hours of exposure. Together, these results showed that girinimbine could effectively suppress angiogenesis, suggestive of its therapeutic potential as a novel angiogenesis inhibitor. Keywords: angiogenesis, inhibitor, carbazole alkaloid, zebrafish

Urokinase receptor expression involves tyrosine phosphorylation of phosphoglycerate kinase.

Science.gov (United States)

Shetty, Praveenkumar; Velusamy, Thirunavukkarasu; Bhandary, Yashodhar P; Liu, Ming C; Shetty, Sreerama

2010-02-01

The interaction of urokinase-type plasminogen activator (uPA) with its receptor, uPAR, plays a central role in several pathophysiological processes, including cancer. uPA induces its own cell surface receptor expression through stabilization of uPAR mRNA. The mechanism involves binding of a 51 nt uPAR mRNA coding sequence with phosphoglycerate kinase (PGK) to down regulate cell surface uPAR expression. Tyrosine phosphorylation of PGK mediated by uPA treatment enhances uPAR mRNA stabilization. In contrast, inhibition of tyrosine phosphorylation augments PGK binding to uPAR mRNA and attenuates uPA-induced uPAR expression. Mapping the specific peptide region of PGK indicated that its first quarter (amino acids 1-100) interacts with uPAR mRNA. To determine if uPAR expression by uPA is regulated through activation of tyrosine residues of PGK, we mutated the specific tyrosine residue and tested mutant PGK for its ability to interfere with uPAR expression. Inhibition of tyrosine phosphorylation by mutating Y76 residue abolished uPAR expression induced by uPA treatment. These findings collectively demonstrate that Y76 residue present in the first quarter of the PGK molecule is involved in lung epithelial cell surface uPAR expression. This region can effectively mimic the function of a whole PGK molecule in inhibiting tumor cell growth.

Bruton tyrosine kinase inhibition in chronic lymphocytic leukemia.

Science.gov (United States)

Maddocks, Kami; Jones, Jeffrey A

2016-04-01

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia and remains incurable outside of the setting of allogeneic stem cell transplant. While the standard therapy for both initial and relapsed CLL has traditionally included monoclonal antibody therapy in combination with chemotherapy, there are patients with high-risk disease features including unmutated IgVH, del(11q22) and del(17p13) that are associated with poor overall responses to these therapies with short time to relapse and shortened overall survival. Additionally, many of these therapies have a high rate of infectious toxicity in a population already at increased risk. Targeting the B-cell receptor (BCR) signaling pathway has emerged as a promising therapeutic advance in a variety of B-cell malignancies, including CLL. Bruton agammaglobulinemia tyrosine kinase (Btk) is a tyrosine kinase in the BCR pathway critical to the survival of both normal and malignant B cells and inhibition of this kinase has shown to block the progression of CLL. Ibrutinib, a first in class oral inhibitor of Btk, has shown promise as a very effective agent in the treatment of CLL-in both relapsed and upfront therapy, alone and in combination with other therapies, and in patients of all-risk disease-which has led to its approval in relapsed CLL and as frontline therapy in patients with the high-risk del(17p13) disease. Several studies are ongoing to evaluate the efficacy and safety of ibrutinib in combination with chemotherapy as frontline treatment for CLL and investigation into newer-generation Btk inhibitors is also underway. Copyright © 2016 Elsevier Inc. All rights reserved.

Acidic pH reduces VEGF-mediated endothelial cell responses by downregulation of VEGFR-2; relevance for anti-angiogenic therapies.

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Faes, Seraina; Uldry, Emilie; Planche, Anne; Santoro, Tania; Pythoud, Catherine; Demartines, Nicolas; Dormond, Olivier

2016-12-27

Anti-angiogenic treatments targeting the vascular endothelial growth factor or its receptors have shown clinical benefits. However, impact on long-term survival remains limited. Solid tumors display an acidic microenvironment that profoundly influences their biology. Consequences of acidity on endothelial cells and anti-angiogenic therapies remain poorly characterized and hence are the focus of this study. We found that exposing endothelial cells to acidic extracellular pH resulted in reduced cell proliferation and migration. Also, whereas VEGF increased endothelial cell proliferation and survival at pH 7.4, it had no effect at pH 6.4. Furthermore, in acidic conditions, stimulation of endothelial cells with VEGF did not result in activation of downstream signaling pathways such as AKT. At a molecular level, acidity significantly decreased the expression of VEGFR-2 by endothelial cells. Consequently, anti-angiogenic therapies that target VEGFR-2 such as sunitinib and sorafenib failed to block endothelial cell proliferation in acidic conditions. In vivo, neutralizing tumor acidity with sodium bicarbonate increased the percentage of endothelial cells expressing VEGFR-2 in tumor xenografts. Furthermore, combining sodium bicarbonate with sunitinib provided stronger anti-cancer activity than either treatment alone. Histological analysis showed that sunitinib had a stronger anti-angiogenic effect when combined with sodium bicarbonate. Overall, our results show that endothelial cells prosper independently of VEGF in acidic conditions partly as a consequence of decreased VEGFR-2 expression. They further suggest that strategies aiming to raise intratumoral pH can improve the efficacy of anti-VEGF treatments.

Tyrosine kinase receptor status in endometrial stromal sarcoma: an immunohistochemical and genetic-molecular analysis.

Science.gov (United States)

Cossu-Rocca, Paolo; Contini, Marcella; Uras, Maria Gabriela; Muroni, Maria Rosaria; Pili, Francesca; Carru, Ciriaco; Bosincu, Luisanna; Massarelli, Giovannino; Nogales, Francisco F; De Miglio, Maria Rosaria

2012-11-01

Endometrial stromal sarcomas (ESS) are rare uterine malignant mesenchymal neoplasms, which are currently treated by surgery, as effective adjuvant therapies have not yet been established. Tyrosine kinase inhibitors have rarely been applied in ESS therapy, with few reports describing imatinib responsivity. The aim of this study was to analyze the status of different tyrosine kinase receptors in an ESS series, in order to evaluate their potential role as molecular targets. Immunohistochemistry was performed for EGFR, c-KIT, PDGFR-α, PDGFR-β, and ABL on 28 ESS. EGFR, PDGFR-α, and PDGFR-β gene expression was investigated by real-time polymerase chain reaction (qRT-PCR) on selected cases. "Hot-spot" mutations were screened for on EGFR, c-KIT, PDGFR-α, and PDGFR-β genes, by sequencing. All analysis was executed from formalin-fixed, paraffin-embedded specimens. Immunohistochemical overexpression of 2 or more tyrosine kinase receptors was observed in 18 of 28 tumors (64%), whereas only 5 tumors were consistently negative. Gene expression profiles were concordant with immunohistochemical overexpression in only 1 tumor, which displayed both high mRNA levels and specific immunoreactivity for PDGFR-α, and PDGFR-β. No activating mutations were found on the tumors included in the study. This study confirms that TKRs expression is frequently observed in ESS. Considering that the responsiveness to tyrosine kinase inhibitors is known to be related to the presence of specific activating mutations or gene over-expression, which are not detectable in ESS, TKRs immunohistochemical over-expression alone should not be considered as a reliable marker for targeted therapies in ESS. Specific post-translational abnormalities, responsible for activation of TKRs, should be further investigated.

Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation

DEFF Research Database (Denmark)

Lotti, L V; Lanfrancone, L; Migliaccio, E

1996-01-01

area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane....... The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein....

Tyrosine Kinase Inhibition in HPV-related Squamous Cell Carcinoma Reveals Beneficial Expression of cKIT and Src.

Science.gov (United States)

Kramer, Benedikt; Kneissle, Marcel; Birk, Richard; Rotter, Nicole; Aderhold, Christoph

2018-05-01

Therapeutic options of locally advanced or metastatic head and neck squamous cell carcinoma (HNSCC) are limited. Src and cKIT are key protein regulators for local tumor progression. The aim of the study was to investigate the therapeutic potential of targeted therapies in human squamous cell carcinoma (HNSCC) in vitro. Therefore, the influence of the selective tyrosine kinase inhibitors niotinib, dasatinib, erlotinib, gefitinib and afatinib on Src and cKIT expression in Human papilloma virus (HPV)-positive and HPV-negative squamous cancer cells (SCC) was analyzed in vitro. ELISA was performed to evaluate the expression of Src and cKIT under the influence of nilotinib, dasatinib, erlotinib, gefitinib and afatinib (10 μmol/l) in HPV-negative and HPV-positive SCC (24-96 h of incubation). Gefitinib significantly increased cKIT expression in HPV-positive and HPV-negative cells whereas nilotinib and afatinib decreased cKIT expression in HPV-positive SCC. The influence of tyrosine kinase inhibitors in HPV-negative SCC was marginal. Surprisingly, Src expression was significantly increased by all tested tyrosine kinase inhibitors in HPV-positive SCC. The results revealed beneficial and unexpected information concerning the interaction of selective tyrosine kinase inhibitors and the tumor biology of HNSCC. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Roles of pro-angiogenic and anti-angiogenic factors as well as matrix metalloproteinases in healing of NSAID-induced small intestinal ulcers in rats.

Science.gov (United States)

Gyenge, Melinda; Amagase, Kikuko; Kunimi, Shino; Matsuoka, Rie; Takeuchi, Koji

2013-10-06

We examined changes in the expression of a pro-angiogenic factor, vascular endothelial growth factor (VEGF), and an anti-angiogenic factor, endostatin, as well as matrix metalloproteinase (MMP)-2 and MMP-9 in the rat small intestine after administration of indomethacin and investigated the roles of these factors in the healing of indomethacin-induced small intestinal ulcers. Male SD rats were given indomethacin (10mg/kg) p.o. and euthanized at various time points (3-24h and 2-7days) after the administration. To impair the healing of these lesions, low-dose of indomethacin (2mg/kg) was given p.o. once daily for 6days starting 1day after ulceration. Levels of VEGF, endostatin, MMP-2 and MMP-9 were determined by Western blotting. The expression of both VEGF and endostatin was upregulated after the ulceration. Repeated administration of low-dose indomethacin impaired the ulcer healing with a decrease of VEGF expression and a further increase of endostatin expression, resulting in a marked decrease in the ratio of VEGF/endostatin expression. The levels of MMP-2 and MMP-9 were both significantly increased after the ulceration, but these responses were suppressed by the repeated indomethacin treatment. The healing of these ulcers was significantly delayed by the repeated administration of MMP inhibitors such as ARP-101 and SB-3CT. The results confirm the importance of the balance between pro-angiogenic and anti-angiogenic activities in the healing of indomethacin-induced small intestinal damage and further suggest that the increased expression of MMP-2 and MMP-9 is another important factor for ulcer healing in the small intestine. © 2013.

A role for the tyrosine kinase ACK1 in neurotrophin signaling and neuronal extension and branching

Science.gov (United States)

La Torre, A; del Mar Masdeu, M; Cotrufo, T; Moubarak, R S; del Río, J A; Comella, J X; Soriano, E; Ureña, J M

2013-01-01

Neurotrophins are involved in many crucial cellular functions, including neurite outgrowth, synapse formation, and plasticity. Although these events have long been known, the molecular determinants underlying neuritogenesis have not been fully characterized. Ack1 (activated Cdc42-associated tyrosine kinase) is a non-receptor tyrosine kinase that is highly expressed in the brain. Here, we demonstrate that Ack1 is a molecular constituent of neurotrophin signaling cascades in neurons and PC12 cells. We report that Ack1 interacts with Trk receptors and becomes tyrosine phosphorylated and its kinase activity is increased in response to neurotrophins. Moreover, our data indicate that Ack1 acts upstream of the Akt and MAPK pathways. We show that Ack1 overexpression induces neuritic outgrowth and promotes branching in neurotrophin-treated neuronal cells, whereas the expression of Ack1 dominant negatives or short-hairpin RNAs counteract neurotrophin-stimulated differentiation. Our results identify Ack1 as a novel regulator of neurotrophin-mediated events in primary neurons and in PC12 cells. PMID:23598414

Ret function in muscle stem cells points to tyrosine kinase inhibitor therapy for facioscapulohumeral muscular dystrophy.

Science.gov (United States)

Moyle, Louise A; Blanc, Eric; Jaka, Oihane; Prueller, Johanna; Banerji, Christopher Rs; Tedesco, Francesco Saverio; Harridge, Stephen Dr; Knight, Robert D; Zammit, Peter S

2016-11-14

Facioscapulohumeral muscular dystrophy (FSHD) involves sporadic expression of DUX4, which inhibits myogenesis and is pro-apoptotic. To identify target genes, we over-expressed DUX4 in myoblasts and found that the receptor tyrosine kinase Ret was significantly up-regulated, suggesting a role in FSHD. RET is dynamically expressed during myogenic progression in mouse and human myoblasts. Constitutive expression of either RET9 or RET51 increased myoblast proliferation, whereas siRNA-mediated knockdown of Ret induced myogenic differentiation. Suppressing RET activity using Sunitinib, a clinically-approved tyrosine kinase inhibitor, rescued differentiation in both DUX4-expressing murine myoblasts and in FSHD patient-derived myoblasts. Importantly, Sunitinib also increased engraftment and differentiation of FSHD myoblasts in regenerating mouse muscle. Thus, DUX4-mediated activation of Ret prevents myogenic differentiation and could contribute to FSHD pathology by preventing satellite cell-mediated repair. Rescue of DUX4-induced pathology by Sunitinib highlights the therapeutic potential of tyrosine kinase inhibitors for treatment of FSHD.

Tyrosine kinase inhibitors and mesenchymal stromal cells: effects on self-renewal, commitment and functions

Science.gov (United States)

Borriello, Adriana; Caldarelli, Ilaria; Bencivenga, Debora; Stampone, Emanuela; Perrotta, Silverio; Oliva, Adriana; Ragione, Fulvio Della

2017-01-01

The hope of selectively targeting cancer cells by therapy and eradicating definitively malignancies is based on the identification of pathways or metabolisms that clearly distinguish “normal” from “transformed” phenotypes. Some tyrosine kinase activities, specifically unregulated and potently activated in malignant cells, might represent important targets of therapy. Consequently, tyrosine kinase inhibitors (TKIs) might be thought as the “vanguard” of molecularly targeted therapy for human neoplasias. Imatinib and the successive generations of inhibitors of Bcr-Abl1 kinase, represent the major successful examples of TKI use in cancer treatment. Other tyrosine kinases have been selected as targets of therapy, but the efficacy of their inhibition, although evident, is less definite. Two major negative effects exist in this therapeutic strategy and are linked to the specificity of the drugs and to the role of the targeted kinase in non-malignant cells. In this review, we will discuss the data available on the TKIs effects on the metabolism and functions of mesenchymal stromal cells (MSCs). MSCs are widely distributed in human tissues and play key physiological roles; nevertheless, they might be responsible for important pathologies. At present, bone marrow (BM) MSCs have been studied in greater detail, for both embryological origins and functions. The available data are evocative of an unexpected degree of complexity and heterogeneity of BM-MSCs. It is conceivable that this grade of intricacy occurs also in MSCs of other organs. Therefore, in perspective, the negative effects of TKIs on MSCs might represent a critical problem in long-term cancer therapies based on such inhibitors. PMID:27750212

A novel spleen tyrosine kinase inhibitor blocks c-Jun N-terminal kinase-mediated gene expression in synoviocytes

NARCIS (Netherlands)

Cha, Hoon-Suk; Boyle, David L.; Inoue, Tomoyuki; Schoot, Reineke; Tak, Paul P.; Pine, Polly; Firestein, Gary S.

2006-01-01

Spleen tyrosine kinase (Syk) is a key regulator of cell signaling induced by cytokines or Fc receptor engagement. However, the role of Syk in rheumatoid arthritis (RA) is not known yet. We investigated the pathways activated by Syk in tumor necrosis factor-alpha (TNFalpha)-stimulated fibroblast-like

Phosphotyrosine phosphatase and tyrosine kinase inhibition modulate airway pressure-induced lung injury.

Science.gov (United States)

Parker, J C; Ivey, C L; Tucker, A

1998-11-01

We determined whether drugs which modulate the state of protein tyrosine phosphorylation could alter the threshold for high airway pressure-induced microvascular injury in isolated perfused rat lungs. Lungs were ventilated for successive 30-min periods with peak inflation pressures (PIP) of 7, 20, 30, and 35 cmH2O followed by measurement of the capillary filtration coefficient (Kfc), a sensitive index of hydraulic conductance. In untreated control lungs, Kfc increased by 1.3- and 3.3-fold relative to baseline (7 cmH2O PIP) after ventilation with 30 and 35 cmH2O PIP. However, in lungs treated with 100 microM phenylarsine oxide (a phosphotyrosine phosphatase inhibitor), Kfc increased by 4.7- and 16.4-fold relative to baseline at these PIP values. In lungs treated with 50 microM genistein (a tyrosine kinase inhibitor), Kfc increased significantly only at 35 cmH2O PIP, and the three groups were significantly different from each other. Thus phosphotyrosine phosphatase inhibition increased the susceptibility of rat lungs to high-PIP injury, and tyrosine kinase inhibition attenuated the injury relative to the high-PIP control lungs.

Angiogenic Factor Profiles in Pregnant Women With a History of Early-Onset Severe Preeclampsia Receiving Low-Molecular-Weight Heparin Prophylaxis.

Science.gov (United States)

Lecarpentier, Edouard; Gris, Jean Christophe; Cochery-Nouvellon, Eva; Mercier, Erick; Touboul, Cyril; Thadhani, Ravi; Karumanchi, S Ananth; Haddad, Bassam

2018-01-01

To evaluate whether daily low-molecular-weight (LMW) heparin prophylaxis during pregnancy alters profile of circulating angiogenic factors that have been linked with the pathogenesis of preeclampsia and fetal growth restriction. This is a planned ancillary study of the Heparin-Preeclampsia trial, a randomized trial in pregnant women with a history of severe early-onset preeclampsia (less than 34 weeks of gestation). In the parent study, all women were treated with aspirin and then randomized to receive LMW heparin or aspirin alone. In this study, we measured serum levels of circulating angiogenic factors (soluble fms-like tyrosine kinase-1, placental growth factor, and soluble endoglin by immunoassay) at the following gestational windows: 10-13 6/7 weeks, 14-17 6/7 weeks, 18-21 6/7 weeks, 22-25 6/7 weeks, 26-29 6/7 weeks, 30-33 6/7 weeks, and 34-37 6/7 weeks. Samples were available from 185 patients: LMW heparin+aspirin (n=92) and aspirin alone (n=93). The two groups had comparable baseline characteristics and had similar adverse composite outcomes (35/92 [38.0%] compared with 36/93 [38.7%]; P=.92). There were no significant differences in serum levels of soluble fms-like tyrosine kinase-1, placental growth factor, and soluble endoglin in the participants who received LMW heparin and aspirin compared with those who received aspirin alone regardless of gestational age period. Finally, women who developed an adverse composite outcome at less than 34 weeks of gestation demonstrated significant alterations in serum angiogenic profile as early as 10-13 6/7 weeks that was most dramatic 6-8 weeks preceding delivery. Prophylactic LMW heparin therapy when beginning from before 14 weeks of gestation with aspirin during pregnancy is not associated with an improved angiogenic profile. This may provide a molecular explanation for the lack of clinical benefit noted in recent trials. ClinicalTrials.gov, NCT00986765.

Review of current classification, molecular alterations, and tyrosine kinase inhibitor therapies in myeloproliferative disorders with hypereosinophilia

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Havelange V

2013-08-01

Full Text Available Violaine Havelange,1,2 Jean-Baptiste Demoulin1 1de Duve Institute, Université catholique de Louvain, Brussels, Belgium; 2Department of Hematology, Cliniques universitaires Saint-Luc, Université catholique de Louvain, Brussels, Belgium Abstract: Recent advances in our understanding of the molecular mechanisms underlying hypereosinophilia have led to the development of a 'molecular' classification of myeloproliferative disorders with eosinophilia. The revised 2008 World Health Organization classification of myeloid neoplasms included a new category called “myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB or FGFR1.” Despite the molecular heterogeneity of PDGFR (platelet-derived growth factor receptor rearrangements, tyrosine kinase inhibitors at low dose induce rapid and complete hematological remission in the majority of these patients. Other kinase inhibitors are promising. Further discoveries of new molecular alterations will direct the development of new specific inhibitors. In this review, an update of the classifications of myeloproliferative disorders associated with hypereosinophilia is discussed together with open and controversial questions. Molecular mechanisms and promising results of tyrosine kinase inhibitor treatments are reviewed. Keywords: hypereosinophilia, classification, myeloproliferative disorders, molecular alterations, tyrosine kinase inhibitor

Tyrosine kinase chromosomal translocations mediate distinct and overlapping gene regulation events

International Nuclear Information System (INIS)

Kim, Hani; Gillis, Lisa C; Jarvis, Jordan D; Yang, Stuart; Huang, Kai; Der, Sandy; Barber, Dwayne L

2011-01-01

Leukemia is a heterogeneous disease commonly associated with recurrent chromosomal translocations that involve tyrosine kinases including BCR-ABL, TEL-PDGFRB and TEL-JAK2. Most studies on the activated tyrosine kinases have focused on proximal signaling events, but little is known about gene transcription regulated by these fusions. Oligonucleotide microarray was performed to compare mRNA changes attributable to BCR-ABL, TEL-PDGFRB and TEL-JAK2 after 1 week of activation of each fusion in Ba/F3 cell lines. Imatinib was used to control the activation of BCR-ABL and TEL-PDGFRB, and TEL-JAK2-mediated gene expression was examined 1 week after Ba/F3-TEL-JAK2 cells were switched to factor-independent conditions. Microarray analysis revealed between 800 to 2000 genes induced or suppressed by two-fold or greater by each tyrosine kinase, with a subset of these genes commonly induced or suppressed among the three fusions. Validation by Quantitative PCR confirmed that eight genes (Dok2, Mrvi1, Isg20, Id1, gp49b, Cxcl10, Scinderin, and collagen Vα1(Col5a1)) displayed an overlapping regulation among the three tested fusion proteins. Stat1 and Gbp1 were induced uniquely by TEL-PDGFRB. Our results suggest that BCR-ABL, TEL-PDGFRB and TEL-JAK2 regulate distinct and overlapping gene transcription profiles. Many of the genes identified are known to be involved in processes associated with leukemogenesis, including cell migration, proliferation and differentiation. This study offers the basis for further work that could lead to an understanding of the specificity of diseases caused by these three chromosomal translocations

An SH2 domain-based tyrosine kinase assay using biotin ligase modified with a terbium(III) complex.

Science.gov (United States)

Sueda, Shinji; Shinboku, Yuki; Kusaba, Takeshi

2013-01-01

Src homology 2 (SH2) domains are modules of approximately 100 amino acids and are known to bind phosphotyrosine-containing sequences with high affinity and specificity. In the present work, we developed an SH2 domain-based assay for Src tyrosine kinase using a unique biotinylation reaction from archaeon Sulfolobus tokodaii. S. tokodaii biotinylation has a unique property that biotin protein ligase (BPL) forms a stable complex with its biotinylated substrate protein (BCCP). Here, an SH2 domain from lymphocyte-specific tyrosine kinase was genetically fused to a truncated BCCP, and the resulting fusion protein was labeled through biotinylation with BPL carrying multiple copies of a luminescent Tb(3+) complex. The labeled SH2 fusion proteins were employed to detect a phosphorylated peptide immobilized on the surface of the microtiter plate, where the phosphorylated peptide was produced by phosphorylation to the substrate peptide by Src tyrosine kinase. Our assay allows for a reliable determination of the activity of Src kinase lower than 10 pg/μL by a simple procedure.

Muscarinic agonists and phorbol esters increase tyrosine phosphorylation of a 40-kilodalton protein in hippocampal slices

International Nuclear Information System (INIS)

Stratton, K.R.; Worley, P.F.; Huganir, R.L.; Baraban, J.M.

1989-01-01

The authors have used the hippocampal slice preparation to investigate the regulation of protein tyrosine phosphorylation in brain. After pharmacological treatment of intact slices, proteins were separated by electrophoresis, and levels of protein tyrosine phosphorylation were assessed by immunoblotting with specific anti-phosphotyrosine antibodies. Phorbol esters, activators of the serine- and threonine-phosphorylating enzyme protein kinase C, selectively increase tyrosine phosphorylation of a soluble protein with an apparent molecular mass of approximately 40 kilodaltons. Muscarinic agonists such as carbachol and oxotremorine M that strongly activate the inositol phospholipid system also increase tyrosine phosphorylation of this protein. Neurotransmitter activation of the inositol phospholipid system and protein kinase C appears to trigger a cascade leading to increased tyrosine phosphorylation

Interactions between Type III receptor tyrosine phosphatases and growth factor receptor tyrosine kinases regulate tracheal tube formation in Drosophila

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Mili Jeon

2012-04-01

The respiratory (tracheal system of the Drosophila melanogaster larva is an intricate branched network of air-filled tubes. Its developmental logic is similar in some ways to that of the vertebrate vascular system. We previously described a unique embryonic tracheal tubulogenesis phenotype caused by loss of both of the Type III receptor tyrosine phosphatases (RPTPs, Ptp4E and Ptp10D. In Ptp4E Ptp10D double mutants, the linear tubes in unicellular and terminal tracheal branches are converted into bubble-like cysts that incorporate apical cell surface markers. This tube geometry phenotype is modulated by changes in the activity or expression of the epidermal growth factor receptor (Egfr tyrosine kinase (TK. Ptp10D physically interacts with Egfr. Here we demonstrate that the Ptp4E Ptp10D phenotype is the consequence of the loss of negative regulation by the RPTPs of three growth factor receptor TKs: Egfr, Breathless and Pvr. Reducing the activity of any of the three kinases by tracheal expression of dominant-negative mutants suppresses cyst formation. By competing dominant-negative and constitutively active kinase mutants against each other, we show that the three RTKs have partially interchangeable activities, so that increasing the activity of one kinase can compensate for the effects of reducing the activity of another. This implies that SH2-domain downstream effectors that are required for the phenotype are likely to be able to interact with phosphotyrosine sites on all three receptor TKs. We also show that the phenotype involves increases in signaling through the MAP kinase and Rho GTPase pathways.

Small Molecule Tyrosine Kinase Inhibitors of ErbB2/HER2/Neu in the Treatment of Aggressive Breast Cancer

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Richard L. Schroeder

2014-09-01

Full Text Available The human epidermal growth factor receptor 2 (HER2 is a member of the erbB class of tyrosine kinase receptors. These proteins are normally expressed at the surface of healthy cells and play critical roles in the signal transduction cascade in a myriad of biochemical pathways responsible for cell growth and differentiation. However, it is widely known that amplification and subsequent overexpression of the HER2 encoding oncogene results in unregulated cell proliferation in an aggressive form of breast cancer known as HER2-positive breast cancer. Existing therapies such as trastuzumab (Herceptin® and lapatinib (Tyverb/Tykerb®, a monoclonal antibody inhibitor and a dual EGFR/HER2 kinase inhibitor, respectively, are currently used in the treatment of HER2-positive cancers, although issues with high recurrence and acquired resistance still remain. Small molecule tyrosine kinase inhibitors provide attractive therapeutic targets, as they are able to block cell signaling associated with many of the proposed mechanisms for HER2 resistance. In this regard we aim to present a review on the available HER2 tyrosine kinase inhibitors, as well as those currently in development. The use of tyrosine kinase inhibitors as sequential or combinatorial therapeutic strategies with other HER family inhibitors is also discussed.

TYROSINE KINASE INHIBITORS AND PREGNANCY

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Elisabetta Abruzzese

2014-04-01

Full Text Available The management of patients with chronic myeloid leukemia (CML during pregnancy has became recently a matter of continuous debate.  The introduction of the Tyrosine Kinase Inhibitors (TKIs in clinical practice has dramatically changed the prognosis of CML patients.  Patients diagnosed in chronic phase can reasonably expect many years of excellent disease control and good quality of life, as well as a normal life expectancy.  This fact has come the necessity to address issues relating to fertility and pregnancy. Physicians are not infrequently being asked for advice regarding the need for, and or the appropriateness of, stopping treatment in order to conceive. In this report we will review the data published in terms of fertility, conception, pregnancy, pregnancy outcome and illness control for all the approved TKIs, as well as suggest how to manage a planned and/or unplanned pregnancy.

The tricarboxylic acid cycle activity in cultured primary astrocytes is strongly accelerated by the protein tyrosine kinase inhibitor tyrphostin 23

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Hohnholt, Michaela C; Blumrich, Eva-Maria; Waagepetersen, Helle S

2017-01-01

production. In addition, T23-treatment strongly increased the molecular carbon labeling of the TCA cycle intermediates citrate, succinate, fumarate and malate, and significantly increased the incorporation of (13)C-labelling into the amino acids glutamate, glutamine and aspartate. These results clearly......Tyrphostin 23 (T23) is a well-known inhibitor of protein tyrosine kinases and has been considered as potential anti-cancer drug. T23 was recently reported to acutely stimulate the glycolytic flux in primary cultured astrocytes. To investigate whether T23 also affects the tricarboxylic acid (TCA...

Defective TCR stimulation in anergized type 2 T helper cells correlates with abrogated p56(lck) and ZAP-70 tyrosine kinase activities.

Science.gov (United States)

Faith, A; Akdis, C A; Akdis, M; Simon, H U; Blaser, K

1997-07-01

Development of IgE-mediated allergic conditions is dependent on the secretion of a Th2 cytokine pattern, including IL-4, IL-5, and IL-13. The induction of anergy would be one mechanism to abrogate cytokine secretion by Th2 cells, which may be pivotal to the allergic response. We demonstrate here that incubation of cloned human CD4+ phospholipase A2 (PLA)-specific Th2 cells with antigenic peptide, in the absence of professional APC, results in a state of nonresponsiveness. The anergic T cells failed to proliferate or secrete IL-4 in response to optimal stimulation with PLA and autologous, professional APC. Secretion of IL-5 and IL-13, however, was only partially inhibited. The anergic state of the Th2 cells was not associated with CD3 or CD28 down-regulation. However, anergy did appear to be closely related to alterations in signaling pathways, mediated through the TCR, of the cells. In contrast to untreated Th2 cells, anergized Th2 cells failed to respond to anti-CD3 mAb with either increased tyrosine kinase activity or increased levels of tyrosine phosphorylation of p56(lck) or ZAP70. A strong and sustained intracellular calcium flux, observed in untreated Th2 cells in response to anti-CD3 mAb, was absent in anergic Th2 cells. Furthermore, the induction of anergy seems to represent an active process, associated with increased levels of basal tyrosine kinase activity, cytokine production, and CD25 up-regulation in anergic Th2 cells. Together, our results indicate that anergy in Th2 cells is associated with defective transmembrane signaling through the TCR.

A protein-tyrosine phosphatase with sequence similarity to the SH2 domain of the protein-tyrosine kinases.

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Shen, S H; Bastien, L; Posner, B I; Chrétien, P

1991-08-22

The phosphorylation of proteins at tyrosine residues is critical in cellular signal transduction, neoplastic transformation and control of the mitotic cycle. These mechanisms are regulated by the activities of both protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPases). As in the PTKs, there are two classes of PTPases: membrane associated, receptor-like enzymes and soluble proteins. Here we report the isolation of a complementary DNA clone encoding a new form of soluble PTPase, PTP1C. The enzyme possesses a large noncatalytic region at the N terminus which unexpectedly contains two adjacent copies of the Src homology region 2 (the SH2 domain) found in various nonreceptor PTKs and other cytoplasmic signalling proteins. As with other SH2 sequences, the SH2 domains of PTP1C formed high-affinity complexes with the activated epidermal growth factor receptor and other phosphotyrosine-containing proteins. These results suggest that the SH2 regions in PTP1C may interact with other cellular components to modulate its own phosphatase activity against interacting substrates. PTPase activity may thus directly link growth factor receptors and other signalling proteins through protein-tyrosine phosphorylation.

VEGFR2 heterogeneity and response to anti-angiogenic low dose metronomic cyclophosphamide treatment

International Nuclear Information System (INIS)

Patten, Steven G; Adamcic, Una; Lacombe, Kristen; Minhas, Kanwal; Skowronski, Karolina; Coomber, Brenda L

2010-01-01

Targeting tumor vasculature is a strategy with great promise in the treatment of many cancers. However, anti-angiogenic reagents that target VEGF/VEGFR2 signaling have met with variable results clinically. Among the possible reasons for this may be heterogeneous expression of the target protein. Double immunofluorescent staining was performed on formalin-fixed paraffin embedded sections of treated and control SW480 (colorectal) and WM239 (melanoma) xenografts, and tissue microarrays of human colorectal carcinoma and melanoma. Xenografts were developed using RAG1 -/- mice by injection with WM239 or SW480 cells and mice were treated with 20 mg/kg/day of cyclophosphamide in their drinking water for up to 18 days. Treated and control tissues were characterized by double immunofluorescence using the mural cell marker α-SMA and CD31, while the ratio of desmin/CD31 was also determined by western blot. Hypoxia in treated and control tissues were quantified using both western blotting for HIF-1α and immunohistochemistry of CA-IX. VEGFR2 is heterogeneously expressed in tumor vasculature in both malignant melanoma and colorectal carcinoma. We observed a significant decrease in microvascular density (MVD) in response to low dose metronomic cyclophosphamide chemotherapy in both malignant melanoma (with higher proportion VEGFR2 positive blood vessels; 93%) and colorectal carcinoma (with lower proportion VEGFR2 positive blood vessels; 60%) xenografts. This reduction in MVD occurred in the absence of a significant anti-tumor effect. We also observed less hypoxia in treated melanoma xenografts, despite successful anti-angiogenic blockade, but no change in hypoxia of colorectal xenografts, suggesting that decreases in tumor hypoxia reflect a complex relationship with vascular density. Based on α-SMA staining and the ratio of desmin to CD31 expression as markers of tumor blood vessel functionality, we found evidence for increased stabilization of colorectal microvessels, but no

In silico design and biological evaluation of a dual specificity kinase inhibitor targeting cell cycle progression and angiogenesis.

Science.gov (United States)

Latham, Antony M; Kankanala, Jayakanth; Fearnley, Gareth W; Gage, Matthew C; Kearney, Mark T; Homer-Vanniasinkam, Shervanthi; Wheatcroft, Stephen B; Fishwick, Colin W G; Ponnambalam, Sreenivasan

2014-01-01

Protein kinases play a central role in tumor progression, regulating fundamental processes such as angiogenesis, proliferation and metastasis. Such enzymes are an increasingly important class of drug target with small molecule kinase inhibitors being a major focus in drug development. However, balancing drug specificity and efficacy is problematic with off-target effects and toxicity issues. We have utilized a rational in silico-based approach to demonstrate the design and study of a novel compound that acts as a dual inhibitor of vascular endothelial growth factor receptor 2 (VEGFR2) and cyclin-dependent kinase 1 (CDK1). This compound acts by simultaneously inhibiting pro-angiogenic signal transduction and cell cycle progression in primary endothelial cells. JK-31 displays potent in vitro activity against recombinant VEGFR2 and CDK1/cyclin B proteins comparable to previously characterized inhibitors. Dual inhibition of the vascular endothelial growth factor A (VEGF-A)-mediated signaling response and CDK1-mediated mitotic entry elicits anti-angiogenic activity both in an endothelial-fibroblast co-culture model and a murine ex vivo model of angiogenesis. We deduce that JK-31 reduces the growth of both human endothelial cells and human breast cancer cells in vitro. This novel synthetic molecule has broad implications for development of similar multi-kinase inhibitors with anti-angiogenic and anti-cancer properties. In silico design is an attractive and innovative method to aid such drug discovery.

Ret receptor tyrosine kinase sustains proliferation and tissue maturation in intestinal epithelia

DEFF Research Database (Denmark)

Perea, Daniel; Guiu, Jordi; Hudry, Bruno

2017-01-01

Expression of the Ret receptor tyrosine kinase is a defining feature of enteric neurons. Its importance is underscored by the effects of its mutation in Hirschsprung disease, leading to absence of gut innervation and severe gastrointestinal symptoms. We report a new and physiologically significant...

Metformin as a prevention and treatment for preeclampsia: effects on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion and endothelial dysfunction.

Science.gov (United States)

Brownfoot, Fiona C; Hastie, Roxanne; Hannan, Natalie J; Cannon, Ping; Tuohey, Laura; Parry, Laura J; Senadheera, Sevvandi; Illanes, Sebastian E; Kaitu'u-Lino, Tu'uhevaha J; Tong, Stephen

2016-03-01

Preeclampsia is associated with placental ischemia/hypoxia and secretion of soluble fms-like tyrosine kinase 1 and soluble endoglin into the maternal circulation. This causes widespread endothelial dysfunction that manifests clinically as hypertension and multisystem organ injury. Recently, small molecule inhibitors of hypoxic inducible factor 1α have been found to reduce soluble fms-like tyrosine kinase 1 and soluble endoglin secretion. However, their safety profile in pregnancy is unknown. Metformin is safe in pregnancy and is also reported to inhibit hypoxic inducible factor 1α by reducing mitochondrial electron transport chain activity. The purposes of this study were to determine (1) the effects of metformin on placental soluble fms-like tyrosine kinase 1 and soluble endoglin secretion, (2) to investigate whether the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion are regulated through the mitochondrial electron transport chain, and (3) to examine its effects on endothelial dysfunction, maternal blood vessel vasodilation, and angiogenesis. We performed functional (in vitro and ex vivo) experiments using primary human tissues to examine the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion from placenta, endothelial cells, and placental villous explants. We used succinate, mitochondrial complex II substrate, to examine whether the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion were mediated through the mitochondria. We also isolated mitochondria from preterm preeclamptic placentas and gestationally matched control subjects and measured mitochondrial electron transport chain activity using kinetic spectrophotometric assays. Endothelial cells or whole maternal vessels were incubated with metformin to determine whether it rescued endothelial dysfunction induced by either tumor necrosis factor-α (to endothelial cells) or placenta villous

Physical and functional interactions between SH2 and SH3 domains of the Src family protein tyrosine kinase p59fyn

NARCIS (Netherlands)

Panchamoorthy, G.; Fukazawa, T.; Stolz, L.; Payne, G.; Reedquist, K.; Shoelson, S.; Songyang, Z.; Cantley, L.; Walsh, C.; Band, H.

1994-01-01

The Src family protein tyrosine kinases participate in signalling through cell surface receptors that lack intrinsic tyrosine kinase domains. All nine members of this family possess adjacent Src homology (SH2 and SH3) domains, both of which are essential for repression of the enzymatic activity. The

Tyrosine Kinase Inhibitors Induced Thyroid Dysfunction: A Review of Its Incidence, Pathophysiology, Clinical Relevance, and Treatment

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Hala Ahmadieh

2013-01-01

Full Text Available Tyrosine kinase inhibitors (TKI belong to a new class of molecular multitargeted anticancer therapy which targets different growth factor receptors and hence attenuates cancer cell survival and growth. Since their introduction as adjunct treatment for renal cell carcinoma and gastrointestinal stromal tumors (GIST, a number of reports have demonstrated that TKI can induce thyroid dysfunction which was especially more common with sunitinib maleate. Many mechanisms with respect to this adverse effect of tyrosine kinase inhibitors have been proposed including their induction of thyroiditis, capillary regression in the thyroid gland, antithyroid peroxidase antibody production, and their ability to decrease iodine uptake by the thyroid gland. Of interest is the observation that TKI-induced thyroid dysfunction may actually be protective as it was shown to improve overall survival, and it was suggested that it may have a prognostic value. Followup on thyroid function tests while patients are maintained on tyrosine kinase inhibitor is strongly recommended. When thyroid dysfunction occurs, appropriate treatment should be individualized depending on patients symptoms and thyroid stimulating hormone level.

Membrane depolarization-induced RhoA/Rho-associated kinase activation and sustained contraction of rat caudal arterial smooth muscle involves genistein-sensitive tyrosine phosphorylation

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Mita, Mitsuo; Tanaka, Hitoshi; Yanagihara, Hayato; Nakagawa, Jun-ichi; Hishinuma, Shigeru; Sutherland, Cindy; Walsh, Michael P.; Shoji, Masaru

2013-01-01

Rho-associated kinase (ROK) activation plays an important role in K+-induced contraction of rat caudal arterial smooth muscle (Mita et al., Biochem J. 2002; 364: 431–40). The present study investigated a potential role for tyrosine kinase activity in K+-induced RhoA activation and contraction. The non-selective tyrosine kinase inhibitor genistein, but not the src family tyrosine kinase inhibitor PP2, inhibited K+-induced sustained contraction (IC50 = 11.3 ± 2.4 µM). Genistein (10 µM) inhibited the K+-induced increase in myosin light chain (LC20) phosphorylation without affecting the Ca2+ transient. The tyrosine phosphatase inhibitor vanadate induced contraction that was reversed by genistein (IC50 = 6.5 ± 2.3 µM) and the ROK inhibitor Y-27632 (IC50 = 0.27 ± 0.04 µM). Vanadate also increased LC20 phosphorylation in a genistein- and Y-27632-dependent manner. K+ stimulation induced translocation of RhoA to the membrane, which was inhibited by genistein. Phosphorylation of MYPT1 (myosin-targeting subunit of myosin light chain phosphatase) was significantly increased at Thr855 and Thr697 by K+ stimulation in a genistein- and Y-27632-sensitive manner. Finally, K+ stimulation induced genistein-sensitive tyrosine phosphorylation of proteins of ∼55, 70 and 113 kDa. We conclude that a genistein-sensitive tyrosine kinase, activated by the membrane depolarization-induced increase in [Ca2+]i, is involved in the RhoA/ROK activation and sustained contraction induced by K+. Ca2+ sensitization, myosin light chain phosphatase, RhoA, Rho-associated kinase, tyrosine kinase PMID:24133693

High Level of Soluble FMS-Like Tyrosine Kinase-1 (sFlt-1 Serum in Pregnancy as a Risk Factor of Preeclampsia

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I Gede Mega Putra

2016-07-01

Full Text Available Background: Recently, etiology and pathogenesis of preeclampsia remain unknown. One of the theory indicating that hypoxia and ischemic placenta caused by abnormal cytotrophoblast invasion in preeclampsia. Soluble Fms-like tyrosine kinase-1 (sFlt-1 serum as a laboratory marker of hypoxia condition that contributes to the occurrence of endothelial damage and clinical manifestations in preeclampsia. Objective: This study was aimed at proving that high level of soluble Fms-like tyrosine kinase-1 (sFlt-1 serum in pregnancy as a risk factor for preeclampsia. Methods: This study was a case control. Among 58 pregnant women studied, 29 women with preeclampsia as a case group and 29 women with normal pregnancy as a control group. Soluble Fms-like tyrosine kinase-1 (sFlt-1 serum was analyzed in the Prodia Laboratory. Collected data were tested for normality using Kolmogorov-Smirnov, then analyzed with independent sample test. Chi-Square test used to determine soluble Fms-like tyrosine kinase-1 (sFlt-1 serum level in preeclampsia. Results: This research concluded that the average level of soluble Fms-like tyrosine kinase-1 (sFlt-1 serum in preeclampsia were 11231.00 ± 8390.3 pg/mL and 3981.62 ± 4921.5 pg/mL in normal pregnancy. Analysis of significance with independent t-test concluded that the value of t = 4.01 and p = 0.001. This mean the average levels of soluble Fms-like tyrosine kinase-1 (sFlt-1serum levels in both groups were difference significantly (p <0.05. Based on the cut-off point of sFlt-1 serum levels was 4505.50 pg/mL with 79.3% sensitivity and 82.8% specificity, the relative risk of preeclampsia was 18 times (OR = 18.40, IK 95% = 4.93 to 68.70, p = 0.001. Conclusion:  Based on this research, high levels of soluble Fms-like tyrosine kinase-1 (sFlt-1 in pregnancy was proved as a risk factor for preeclampsia.

Effect of tyrosine kinase blockade on norepinephrine-induced cytosolic calcium response in rat afferent arterioles

DEFF Research Database (Denmark)

Salomonsson, Max; Arendshorst, William J

2004-01-01

We used genistein (Gen) and tyrphostin 23 (Tyr-23) to evaluate the importance of tyrosine phosphorylation in norepinephrine (NE)-induced changes in intracellular free calcium concentration ([Ca(2+)](i)) in rat afferent arterioles. [Ca(2+)](i) was measured in microdissected arterioles using...... ratiometric photometry of fura 2 fluorescence. The control [Ca(2+)](i) response to NE (1 microM) consisted of a rapid initial peak followed by a plateau phase sustained above baseline. Pretreatment with the tyrosine kinase inhibitor Tyr-23 (50 microM, 10 min) caused a slow 40% increase in baseline [Ca(2+)](i...... of nifedipine and Tyr-23 were not additive. Nifedipine had no inhibitory effect after Tyr-23 pretreatment, indicating Tyr-23 inhibition of Ca(2+) entry. Another tyrosine kinase inhibitor, Gen (5 and 50 microM), did not affect baseline [Ca(2+)](i). High-dose Gen inhibited the peak and plateau response to NE...

The protist, Monosiga brevicollis, has a tyrosine kinase signaling network more elaborate and diverse than found in any known metazoan.

Science.gov (United States)

Manning, Gerard; Young, Susan L; Miller, W Todd; Zhai, Yufeng

2008-07-15

Tyrosine kinase signaling has long been considered a hallmark of intercellular communication, unique to multicellular animals. Our genomic analysis of the unicellular choanoflagellate Monosiga brevicollis discovers a remarkable count of 128 tyrosine kinases, 38 tyrosine phosphatases, and 123 phosphotyrosine (pTyr)-binding SH2 proteins, all higher counts than seen in any metazoan. This elaborate signaling network shows little orthology to metazoan counterparts yet displays many innovations reminiscent of metazoans. These include extracellular domains structurally related to those of metazoan receptor kinases, alternative methods for membrane anchoring and phosphotyrosine interaction in cytoplasmic kinases, and domain combinations that link kinases to small GTPase signaling and transcription. These proteins also display a wealth of combinations of known signaling domains. This uniquely divergent and elaborate signaling network illuminates the early evolution of pTyr signaling, explores innovative ways to traverse the cellular signaling circuitry, and shows extensive convergent evolution, highlighting pervasive constraints on pTyr signaling.

Protein-tyrosine phosphatase SHP2 contributes to GDNF neurotrophic activity through direct binding to phospho-Tyr687 in the RET receptor tyrosine kinase.

Science.gov (United States)

Perrinjaquet, Maurice; Vilar, Marçal; Ibáñez, Carlos F

2010-10-08

The signaling mechanisms by which neurotrophic receptors regulate neuronal survival and axonal growth are still incompletely understood. In the receptor tyrosine kinase RET, a receptor for GDNF (glial cell line-derived neurotrophic factor), the functions of the majority of tyrosine residues that become phosphorylated are still unknown. Here we have identified the protein-tyrosine phosphatase SHP2 as a novel direct interactor of RET and the first effector known to bind to phosphorylated Tyr(687) in the juxtamembrane region of the receptor. We show that SHP2 is recruited to RET upon ligand binding in a cooperative fashion, such that both interaction with Tyr(687) and association with components of the Tyr(1062) signaling complex are required for stable recruitment of SHP2 to the receptor. SHP2 recruitment contributes to the ability of RET to activate the PI3K/AKT pathway and promote survival and neurite outgrowth in primary neurons. Furthermore, we find that activation of protein kinase A (PKA) by forskolin reduces the recruitment of SHP2 to RET and negatively affects ligand-mediated neurite outgrowth. In agreement with this, mutation of Ser(696), a known PKA phosphorylation site in RET, enhances SHP2 binding to the receptor and eliminates the effect of forskolin on ligand-induced outgrowth. Together, these findings establish SHP2 as a novel positive regulator of the neurotrophic activities of RET and reveal Tyr(687) as a critical platform for integration of RET and PKA signals. We anticipate that several other phosphotyrosines of unknown function in neuronal receptor tyrosine kinases will also support similar regulatory functions.

Anti-angiogenic and cytotoxicity studies of some medicinal plants.

Science.gov (United States)

Ng, Kwok-Wen; Salhimi, Salizawati Muhamad; Majid, Amin Malik; Chan, Kit-Lam

2010-06-01

Angiogenesis plays an important role in tumor formation and proliferation. The development of anti-angiogenic agents to block new blood vessel growth will inhibit metastasis and induce apoptosis of the cancer cells. Nine medicinal plants, Strobilanthes crispus, Phyllanthus niruri, Phyllanthus pulcher, Phyllanthus urinaria, Ailanthus malabarica, Irvingia malayana, Smilax myosotiflora, Tinospora crispa and blumea balsamifera were screened for anti-angiogenic properties using the rat aortic ring assay. Of these, the methanol extracts of Phyllanthus species and Irvingia malayana exhibited the highest activity. At 100 microg/mL, P. pulcher, P. niruri, P. urinaria and I. malayana recorded an inhibition of 78.8 %, 59.5 %, 56.7 % and 46.4 %, respectively, against rat aortic vascular growth. Their activities were further investigated by the tube formation assay involving human umbilical vein endothelial cells (HUVEC) on Matrigel. I. malayana, P. niruri and P. urinaria showed a significant decrease of 45.5, 37.9 and 35.6 %, respectively, whilst P. pulcher showed a much lower decrease of 15.5 % when compared with that of the rat aortic ring assay. All the plant extracts were evaluated for cytotoxicity on a panel of human cancer cell lines using the MTT assay. None of them displayed acute cytotoxicity. The HPLC of P. niruri, P. urinaria and P. pulcher indicated the extracts contained some identical chromatographic peaks of lignans. Further fractionation of I. malayana yielded betulinic acid reported in this plant for the first time and at 100 microg/mL it exhibited a 67.3 % inhibition of vessel outgrowth and 46.5 % inhibition of tube formation. Georg Thieme Verlag KG Stuttgart-New York.

ABL tyrosine kinase inhibition variable effects on the invasive properties of different triple negative breast cancer cell lines.

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Clément Chevalier

Full Text Available The non-receptor tyrosine kinase ABL drives myeloid progenitor expansion in human chronic myeloid leukemia. ABL inhibition by the tyrosine kinase inhibitor nilotinib is a first-line treatment for this disease. Recently, ABL has also been implicated in the transforming properties of solid tumors, including triple negative (TN breast cancer. TN breast cancers are highly metastatic and several cell lines derived from these tumors display high invasive activity in vitro. This feature is associated with the activation of actin-rich membrane structures called invadopodia that promote extracellular matrix degradation. Here, we investigated nilotinib effect on the invasive and migratory properties of different TN breast cancer cell lines. Nilotinib decreased both matrix degradation and invasion in the TN breast cancer cell lines MDA-MB 231 and MDA-MB 468. However, and unexpectedly, nilotinib increased by two-fold the invasive properties of the TN breast cancer cell line BT-549 and of Src-transformed fibroblasts. Both display much higher levels of ABL kinase activity compared to MDA-MB 231. Similar effects were obtained by siRNA-mediated down-regulation of ABL expression, confirming ABL central role in this process. ABL anti-tumor effect in BT-549 cells and Src-transformed fibroblasts was not dependent on EGF secretion, as recently reported in neck and squamous carcinoma cells. Rather, we identified the TRIO-RAC1 axis as an important downstream element of ABL activity in these cancer cells. In conclusion, the observation that TN breast cancer cell lines respond differently to ABL inhibitors could have implications for future therapies.

How tyrosine kinase inhibitors impair metabolism and endocrine system function: a systematic updated review.

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Breccia, Massimo; Molica, Matteo; Alimena, Giuliana

2014-12-01

Tyrosine kinase inhibitors (TKIs) advent has deeply changed the outcome of chronic myeloid leukemia (CML) patients, with improved rates of response and overall survival. However, for this success some patients paid the price of a number of peculiar side effects, the so-called off-target side effects, specific for each one TKI. These effects are due to non-selective inhibition of other tyrosine kinase receptors, such as PDGFR, c-KIT, Src, VEGF. Consequences of this inhibition, some metabolic changes during the treatment with TKIs are reported. Aim of present review is to report metabolic changes and potential mechanisms involved in the pathogenesis related to imatinib, second (nilotinib and dasatinib) and third generation (bosutinib and ponatinib) TKIs. Copyright © 2014 Elsevier Ltd. All rights reserved.

ROLE OF TYROSINE-SULFATED PROTEINS IN RETINAL STRUCTURE AND FUNCTION

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Kanan, Y.; Al-Ubaidi, M.R.

2014-01-01

The extracellular matrix (ECM) plays a significant role in cellular and retinal health. The study of retinal tyrosine-sulfated proteins is an important first step toward understanding the role of ECM in retinal health and diseases. These secreted proteins are members of the retinal ECM. Tyrosine sulfation was shown to be necessary for the development of proper retinal structure and function. The importance of tyrosine sulfation is further demonstrated by the evolutionary presence of tyrosylprotein sulfotransferases, enzymes that catalyze proteins’ tyrosine sulfation, and the compensatory abilities of these enzymes. Research has identified four tyrosine-sulfated retinal proteins: fibulin 2, vitronectin, complement factor H (CFH), and opticin. Vitronectin and CFH regulate the activation of the complement system and are involved in the etiology of some cases of age-related macular degeneration. Analysis of the role of tyrosine sulfation in fibulin function showed that sulfation influences the protein's ability to regulate growth and migration. Although opticin was recently shown to exhibit anti-angiogenic properties, it is not yet determined what role sulfation plays in that function. Future studies focusing on identifying all of the tyrosine-sulfated retinal proteins would be instrumental in determining the impact of sulfation on retinal protein function in retinal homeostasis and diseases. PMID:25819460

Downstream of tyrosine kinase/docking protein 6, as a novel substrate of tropomyosin-related kinase C receptor, is involved in neurotrophin 3-mediated neurite outgrowth in mouse cortex neurons

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Yuan Jian

2010-06-01

Full Text Available Abstract Background The downstream of tyrosine kinase/docking protein (Dok adaptor protein family has seven members, Dok1 to Dok7, that act as substrates of multiple receptor tyrosine kinase and non-receptor tyrosine kinase. The tropomyosin-related kinase (Trk receptor family, which has three members (TrkA, TrkB and TrkC, are receptor tyrosine kinases that play pivotal roles in many stages of nervous system development, such as differentiation, migration, axon and dendrite projection and neuron patterning. Upon related neurotrophin growth factor stimulation, dimerisation and autophosphorylation of Trk receptors can occur, recruiting adaptor proteins to mediate signal transduction. Results In this report, by using yeast two-hybrid assays, glutathione S-transferase (GST precipitation assays and coimmunoprecipitation (Co-IP experiments, we demonstrate that Dok6 selectively binds to the NPQY motif of TrkC through its phosphotyrosine-binding (PTB domain in a kinase activity-dependent manner. We further confirmed their interaction by coimmunoprecipitation and colocalisation in E18.5 mouse cortex neurons, which provided more in vivo evidence. Next, we demonstrated that Dok6 is involved in neurite outgrowth in mouse cortex neurons via the RNAi method. Knockdown of Dok6 decreased neurite outgrowth in cortical neurons upon neurotrophin 3 (NT-3 stimulation. Conclusions We conclude that Dok6 interacts with the NPQY motif of the TrkC receptor through its PTB domain in a kinase activity-dependent manner, and works as a novel substrate of the TrkC receptor involved in NT-3-mediated neurite outgrowth in mouse cortex neurons.

Dialkoxyquinazolines: Screening Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors for Potential Tumor Imaging Probes

International Nuclear Information System (INIS)

VanBrocklin, Henry F.; Lim, John K.; Coffing, Stephanie L.; Hom, Darren L.; Negash, Kitaw; Ono, Michele Y.; Hanrahan, Stephen M.; Taylor, Scott E.; Vanderpoel, Jennifer L.; Slavik, Sarah M.; Morris, Andrew B.; Riese II, David J.

2005-01-01

The epidermal growth factor receptor (EGFR), a long-standing drug development target, is also a desirable target for imaging. Sixteen dialkoxyquinazoline analogs, suitable for labeling with positron-emitting isotopes, have been synthesized and evaluated in a battery of in vitro assays to ascertain their chemical and biological properties. These characteristics provided the basis for the adoption of a selection schema to identify lead molecules for labeling and in vivo evaluation. A newEGFR tyrosine kinase radiometric binding assay revealed that all of the compounds possessed suitable affinity (IC50 = 0.4 - 51 nM) for the EGFR tyrosine kinase. All of the analogs inhibited ligand-induced EGFR tyrosine phosphorylation (IC50 = 0.8 - 20 nM). The HPLC-estimated octanol/water partition coefficients ranged from 2.0-5.5. Four compounds,4-(2'-fluoroanilino)- and 4-(3'-fluoroanilino)-6,7-diethoxyquinazoline as well as 4-(3'-chloroanilino)- and4-(3'-bromoanilino)-6,7-dimethoxyquinazoline, possess the best combination of characteristics that warrant radioisotope labeling and further evaluation in tumor-bearing mice

Multi-lobulation of the nucleus in prolonged S phase by nuclear expression of Chk tyrosine kinase.

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Nakayama, Yuji; Yamaguchi, Naoto

2005-04-01

Chk tyrosine kinase phosphorylates Src-family tyrosine kinases and suppresses their kinase activity. We recently showed that Chk localizes to the nucleus as well as the cytoplasm and inhibits cell proliferation. To investigate the role of nuclear Chk in proliferation, various Chk mutants were constructed and expressed. Nuclear localization of Chk-induced dynamic multi-lobulation of the nucleus and prolonged S phase of the cell cycle. The N-terminal domain of Chk and a portion of its kinase domain but not the kinase activity were responsible for induction of the multi-lobulation. Cell sorting analysis revealed that nuclear multi-lobulated cells were enriched in late S phase. Multi-lobulated nuclei were surrounded with lamin B1 that was particularly concentrated in concave regions of the nuclei. Furthermore, treatment with nocodazole or taxol disrupted multi-lobulation of the nucleus. These results suggest that nuclear multi-lobulation in late S phase, which is dependent on polymerization and depolymerization of microtubules, may be involved in nuclear Chk-induced inhibition of proliferation.

Multi-lobulation of the nucleus in prolonged S phase by nuclear expression of Chk tyrosine kinase

International Nuclear Information System (INIS)

Nakayama, Yuji; Yamaguchi, Naoto

2005-01-01

Chk tyrosine kinase phosphorylates Src-family tyrosine kinases and suppresses their kinase activity. We recently showed that Chk localizes to the nucleus as well as the cytoplasm and inhibits cell proliferation. To investigate the role of nuclear Chk in proliferation, various Chk mutants were constructed and expressed. Nuclear localization of Chk-induced dynamic multi-lobulation of the nucleus and prolonged S phase of the cell cycle. The N-terminal domain of Chk and a portion of its kinase domain but not the kinase activity were responsible for induction of the multi-lobulation. Cell sorting analysis revealed that nuclear multi-lobulated cells were enriched in late S phase. Multi-lobulated nuclei were surrounded with lamin B1 that was particularly concentrated in concave regions of the nuclei. Furthermore, treatment with nocodazole or taxol disrupted multi-lobulation of the nucleus. These results suggest that nuclear multi-lobulation in late S phase, which is dependent on polymerization and depolymerization of microtubules, may be involved in nuclear Chk-induced inhibition of proliferation

Icotinib, a potent and specific EGFR tyrosine kinase inhibitor, inhibits growth of squamous cell carcinoma cell line A431 through negatively regulating AKT signaling.

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Gao, Zhenzhen; Chen, Wei; Zhang, Xiaohua; Cai, Peifen; Fang, Xianying; Xu, Qiang; Sun, Yang; Gu, Yanhong

2013-06-01

Icotinib is a potent and specific epidermal growth factor receptor tyrosine kinase inhibitor. In this study, we reported that icotinib had the antitumor activity on human squamous cell carcinoma cell line A431 in vitro. Meanwhile, adhesion to fibronectin and expression of integrin α3 and β1 were significantly reduced in a dose-dependent manner after the treatment of icotinib. Moreover, icotinib induced cell cycle arrested and affected expression of various cell cycle related proteins in squamous cancer cell line A431, whereas it did not cause apoptosis. Furthermore, icotinib remarkably down-regulated phosphorylation of protein kinase B (AKT) though blocking the interaction between 3-phosphoinositide-dependent protein kinase-1 (PDK1) and AKT in A431 cells. Taken together, it is shown that the small molecular compound, icotinib, has an anti-squamous cell carcinoma activity in vitro and its antitumor mechanism is associated with the blockage of the interaction between PDK1 and AKT. These results provide a novel strategy for anti-squamous cell carcinoma therapy. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Tyrosine kinases, drugs, and Shigella flexneri dissemination.

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Dragoi, Ana-Maria; Agaisse, Hervé

2014-01-01

Shigella flexneri is an enteropathogenic bacterium responsible for approximately 100 million cases of severe dysentery each year. S. flexneri colonization of the human colonic epithelium is supported by direct spread from cell to cell, which relies on actin-based motility. We have recently uncovered that, in intestinal epithelial cells, S. flexneri actin-based motility is regulated by the Bruton's tyrosine kinase (Btk). Consequently, treatment with Ibrutinib, a specific Btk inhibitor currently used in the treatment of B-cell malignancies, effectively impaired S. flexneri spread from cell to cell. Thus, therapeutic intervention capitalizing on drugs interfering with host factors supporting the infection process may represent an effective alternative to treatments with antimicrobial compounds.

TAM receptor tyrosine kinases as emerging targets of innate immune checkpoint blockade for cancer therapy.

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Akalu, Yemsratch T; Rothlin, Carla V; Ghosh, Sourav

2017-03-01

Cancer immunotherapy utilizing T-cell checkpoint inhibitors has shown tremendous clinical success. Yet, this mode of treatment is effective in only a subset of patients. Unresponsive patients tend to have non-T-cell-inflamed tumors that lack markers associated with the activation of adaptive anti-tumor immune responses. Notably, elimination of cancer cells by T cells is critically dependent on the optimal activity of innate immune cells. Therefore, identifying new targets that regulate innate immune cell function and promote the engagement of adaptive tumoricidal responses is likely to lead to the development of improved therapies against cancer. Here, we review the TAM receptor tyrosine kinases-TYRO3, AXL, and MERTK-as an emerging class of innate immune checkpoints that participate in key steps of anti-tumoral immunity. Namely, TAM-mediated efferocytosis, negative regulation of dendritic cell activity, and dysregulated production of chemokines collectively favor the escape of malignant cells. Hence, disabling TAM signaling may promote engagement of adaptive immunity and complement T-cell checkpoint blockade. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Protein-tyrosine Phosphatase SHP2 Contributes to GDNF Neurotrophic Activity through Direct Binding to Phospho-Tyr687 in the RET Receptor Tyrosine Kinase*

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Perrinjaquet, Maurice; Vilar, Marçal; Ibáñez, Carlos F.

2010-01-01

The signaling mechanisms by which neurotrophic receptors regulate neuronal survival and axonal growth are still incompletely understood. In the receptor tyrosine kinase RET, a receptor for GDNF (glial cell line-derived neurotrophic factor), the functions of the majority of tyrosine residues that become phosphorylated are still unknown. Here we have identified the protein-tyrosine phosphatase SHP2 as a novel direct interactor of RET and the first effector known to bind to phosphorylated Tyr687 in the juxtamembrane region of the receptor. We show that SHP2 is recruited to RET upon ligand binding in a cooperative fashion, such that both interaction with Tyr687 and association with components of the Tyr1062 signaling complex are required for stable recruitment of SHP2 to the receptor. SHP2 recruitment contributes to the ability of RET to activate the PI3K/AKT pathway and promote survival and neurite outgrowth in primary neurons. Furthermore, we find that activation of protein kinase A (PKA) by forskolin reduces the recruitment of SHP2 to RET and negatively affects ligand-mediated neurite outgrowth. In agreement with this, mutation of Ser696, a known PKA phosphorylation site in RET, enhances SHP2 binding to the receptor and eliminates the effect of forskolin on ligand-induced outgrowth. Together, these findings establish SHP2 as a novel positive regulator of the neurotrophic activities of RET and reveal Tyr687 as a critical platform for integration of RET and PKA signals. We anticipate that several other phosphotyrosines of unknown function in neuronal receptor tyrosine kinases will also support similar regulatory functions. PMID:20682772

The role of Ryk and Ror receptor tyrosine kinases in Wnt signal transduction

NARCIS (Netherlands)

Green, J.; Nusse, R.; van Amerongen, R.

2014-01-01

Receptor tyrosine kinases of the Ryk and Ror families were initially classified as orphan receptors because their ligands were unknown. They are now known to contain functional extracellular Wnt-binding domains and are implicated in Wnt-signal transduction in multiple species. Although their

Identification of a functional interaction between Kv4.3 channels and c-Src tyrosine kinase.

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Gomes, Pedro; Saito, Tomoaki; Del Corsso, Cris; Alioua, Abderrahmane; Eghbali, Mansoureh; Toro, Ligia; Stefani, Enrico

2008-10-01

Voltage-gated K(+) (Kv) channels are key determinants of cardiac and neuronal excitability. A substantial body of evidence has accumulated in support of a role for Src family tyrosine kinases in the regulation of Kv channels. In this study, we examined the possibility that c-Src tyrosine kinase participates in the modulation of the transient voltage-dependent K(+) channel Kv4.3. Supporting a mechanistic link between Kv4.3 and c-Src, confocal microscopy analysis of HEK293 cells stably transfected with Kv4.3 showed high degree of co-localization of the two proteins at the plasma membrane. Our results further demonstrate an association between Kv4.3 and c-Src by co-immunoprecipitation and GST pull-down assays, this interaction being mediated by the SH2 and SH3 domains of c-Src. Furthermore, we show that Kv4.3 is tyrosine phosphorylated under basal conditions. The functional relevance of the observed interaction between Kv4.3 and c-Src was established in patch-clamp experiments, where application of the Src inhibitor PP2 caused a decrease in Kv4.3 peak current amplitude, but not the inactive structural analogue PP3. Conversely, intracellular application of recombinant c-Src kinase or the protein tyrosine phosphatase inhibitor bpV(phen) increased Kv4.3 peak current amplitude. In conclusion, our findings provide evidence that c-Src-induced Kv4.3 channel activation involves their association in a macromolecular complex and suggest a role for c-Src-Kv4.3 pathway in regulating cardiac and neuronal excitability.

VEGFR2 heterogeneity and response to anti-angiogenic low dose metronomic cyclophosphamide treatment

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Skowronski Karolina

2010-12-01

Full Text Available Abstract Background Targeting tumor vasculature is a strategy with great promise in the treatment of many cancers. However, anti-angiogenic reagents that target VEGF/VEGFR2 signaling have met with variable results clinically. Among the possible reasons for this may be heterogeneous expression of the target protein. Methods Double immunofluorescent staining was performed on formalin-fixed paraffin embedded sections of treated and control SW480 (colorectal and WM239 (melanoma xenografts, and tissue microarrays of human colorectal carcinoma and melanoma. Xenografts were developed using RAG1-/- mice by injection with WM239 or SW480 cells and mice were treated with 20 mg/kg/day of cyclophosphamide in their drinking water for up to 18 days. Treated and control tissues were characterized by double immunofluorescence using the mural cell marker α-SMA and CD31, while the ratio of desmin/CD31 was also determined by western blot. Hypoxia in treated and control tissues were quantified using both western blotting for HIF-1α and immunohistochemistry of CA-IX. Results VEGFR2 is heterogeneously expressed in tumor vasculature in both malignant melanoma and colorectal carcinoma. We observed a significant decrease in microvascular density (MVD in response to low dose metronomic cyclophosphamide chemotherapy in both malignant melanoma (with higher proportion VEGFR2 positive blood vessels; 93% and colorectal carcinoma (with lower proportion VEGFR2 positive blood vessels; 60% xenografts. This reduction in MVD occurred in the absence of a significant anti-tumor effect. We also observed less hypoxia in treated melanoma xenografts, despite successful anti-angiogenic blockade, but no change in hypoxia of colorectal xenografts, suggesting that decreases in tumor hypoxia reflect a complex relationship with vascular density. Based on α-SMA staining and the ratio of desmin to CD31 expression as markers of tumor blood vessel functionality, we found evidence for increased

Quantitative Tyrosine Phosphoproteomics of Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor-treated Lung Adenocarcinoma Cells Reveals Potential Novel Biomarkers of Therapeutic Response.

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Zhang, Xu; Maity, Tapan; Kashyap, Manoj K; Bansal, Mukesh; Venugopalan, Abhilash; Singh, Sahib; Awasthi, Shivangi; Marimuthu, Arivusudar; Charles Jacob, Harrys Kishore; Belkina, Natalya; Pitts, Stephanie; Cultraro, Constance M; Gao, Shaojian; Kirkali, Guldal; Biswas, Romi; Chaerkady, Raghothama; Califano, Andrea; Pandey, Akhilesh; Guha, Udayan

2017-05-01

Mutations in the Epidermal growth factor receptor (EGFR) kinase domain, such as the L858R missense mutation and deletions spanning the conserved sequence 747 LREA 750 , are sensitive to tyrosine kinase inhibitors (TKIs). The gatekeeper site residue mutation, T790M accounts for around 60% of acquired resistance to EGFR TKIs. The first generation EGFR TKIs, erlotinib and gefitinib, and the second generation inhibitor, afatinib are FDA approved for initial treatment of EGFR mutated lung adenocarcinoma. The predominant biomarker of EGFR TKI responsiveness is the presence of EGFR TKI-sensitizing mutations. However, 30-40% of patients with EGFR mutations exhibit primary resistance to these TKIs, underscoring the unmet need of identifying additional biomarkers of treatment response. Here, we sought to characterize the dynamics of tyrosine phosphorylation upon EGFR TKI treatment of mutant EGFR-driven human lung adenocarcinoma cell lines with varying sensitivity to EGFR TKIs, erlotinib and afatinib. We employed stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative mass spectrometry to identify and quantify tyrosine phosphorylated peptides. The proportion of tyrosine phosphorylated sites that had reduced phosphorylation upon erlotinib or afatinib treatment correlated with the degree of TKI-sensitivity. Afatinib, an irreversible EGFR TKI, more effectively inhibited tyrosine phosphorylation of a majority of the substrates. The phosphosites with phosphorylation SILAC ratios that correlated with the TKI-sensitivity of the cell lines include sites on kinases, such as EGFR-Y1197 and MAPK7-Y221, and adaptor proteins, such as SHC1-Y349/350, ERRFI1-Y394, GAB1-Y689, STAT5A-Y694, DLG3-Y705, and DAPP1-Y139, suggesting these are potential biomarkers of TKI sensitivity. DAPP1, is a novel target of mutant EGFR signaling and Y-139 is the major site of DAPP1 tyrosine phosphorylation. We also uncovered several off-target effects of these TKIs, such as MST1R-Y1238

In silico design and biological evaluation of a dual specificity kinase inhibitor targeting cell cycle progression and angiogenesis.

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Antony M Latham

Full Text Available Protein kinases play a central role in tumor progression, regulating fundamental processes such as angiogenesis, proliferation and metastasis. Such enzymes are an increasingly important class of drug target with small molecule kinase inhibitors being a major focus in drug development. However, balancing drug specificity and efficacy is problematic with off-target effects and toxicity issues.We have utilized a rational in silico-based approach to demonstrate the design and study of a novel compound that acts as a dual inhibitor of vascular endothelial growth factor receptor 2 (VEGFR2 and cyclin-dependent kinase 1 (CDK1. This compound acts by simultaneously inhibiting pro-angiogenic signal transduction and cell cycle progression in primary endothelial cells. JK-31 displays potent in vitro activity against recombinant VEGFR2 and CDK1/cyclin B proteins comparable to previously characterized inhibitors. Dual inhibition of the vascular endothelial growth factor A (VEGF-A-mediated signaling response and CDK1-mediated mitotic entry elicits anti-angiogenic activity both in an endothelial-fibroblast co-culture model and a murine ex vivo model of angiogenesis.We deduce that JK-31 reduces the growth of both human endothelial cells and human breast cancer cells in vitro. This novel synthetic molecule has broad implications for development of similar multi-kinase inhibitors with anti-angiogenic and anti-cancer properties. In silico design is an attractive and innovative method to aid such drug discovery.

Fibrocytes: A Novel Stromal Cells to Regulate Resistance to Anti-Angiogenic Therapy and Cancer Progression.

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Goto, Hisatsugu; Nishioka, Yasuhiko

2017-12-29

An adequate blood supply is essential for cancer cells to survive and grow; thus, the concept of inhibiting tumor angiogenesis has been applied to cancer therapy, and several drugs are already in clinical use. It has been shown that treatment with those anti-angiogenic drugs improved the response rate and prolonged the survival of patients with various types of cancer; however, it is also true that the effect was mostly limited. Currently, the disappointing clinical results are explained by the existence of intrinsic or acquired resistance to the therapy mediated by both tumor cells and stromal cells. This article reviews the mechanisms of resistance mediated by stromal cells such as endothelial cells, pericytes, fibroblasts and myeloid cells, with an emphasis on fibrocytes, which were recently identified as the cell type responsible for regulating acquired resistance to anti-angiogenic therapy. In addition, the other emerging role of fibrocytes as mediator-producing cells in tumor progression is discussed.

Molecular features of interaction between VEGFA and anti-angiogenic drugs used in retinal diseases: a computational approach

Directory of Open Access Journals (Sweden)

Chiara Bianca Maria Platania

2015-10-01

Full Text Available Anti-angiogenic agents are biological drugs used for treatment of retinal neovascular degenerative diseases. In this study, we aimed at in-silico analysis of interaction of vascular endothelial growth factor A (VEGFA, the main mediator of angiogenesis, with binding domains of anti-angiogenic agents used for treatment of retinal diseases, such as ranibizumab, bevacizumab and aflibercept. The analysis of anti-VEGF/VEGFA complexes was carried out by means of protein-protein docking and molecular dynamics (MD coupled to molecular mechanics-Poisson Boltzmann Surface Area (MM-PBSA calculation. Molecular dynamics simulation was further analyzed by protein contact networks. Rough energetic evaluation with protein-protein docking scores revealed that aflibercept/VEGFA complex was characterized by electrostatic stabilization, whereas ranibizumab and bevacizumab complexes were stabilized by Van der Waals (VdW energy term; these results were confirmed by MM-PBSA. Comparison of MM-PBSA predicted energy terms with experimental binding parameters reported in literature indicated that the high association rate (Kon of aflibercept to VEGFA was consistent with high stabilizing electrostatic energy. On the other hand, the relatively low experimental dissociation rate (Koff of ranibizumab may be attributed to lower conformational fluctuations of the ranibizumab/VEGFA complex, higher number of contacts and hydrogen bonds in comparison to bevacizumab and aflibercept. Thus, the anti-angiogenic agents have been found to be considerably different both in terms of molecular interactions and stabilizing energy. Characterization of such features can improve the design of novel biological drugs potentially useful in clinical practice.

Small tyrosine kinase inhibitors interrupt EGFR signaling by interacting with erbB3 and erbB4 in glioblastoma cell lines

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Carrasco-Garcia, Estefania; Saceda, Miguel [Instituto de Biologia Molecular y Celular, Universidad Miguel Hernandez, 03202 Elche (Alicante) (Spain); Unidad de Investigacion, Hospital General Universitario de Elche, 03203 Elche (Alicante) (Spain); Grasso, Silvina; Rocamora-Reverte, Lourdes; Conde, Mariano; Gomez-Martinez, Angeles [Instituto de Biologia Molecular y Celular, Universidad Miguel Hernandez, 03202 Elche (Alicante) (Spain); Garcia-Morales, Pilar [Instituto de Biologia Molecular y Celular, Universidad Miguel Hernandez, 03202 Elche (Alicante) (Spain); Unidad de Investigacion, Hospital General Universitario de Elche, 03203 Elche (Alicante) (Spain); Ferragut, Jose A. [Instituto de Biologia Molecular y Celular, Universidad Miguel Hernandez, 03202 Elche (Alicante) (Spain); Martinez-Lacaci, Isabel, E-mail: imlacaci@umh.es [Instituto de Biologia Molecular y Celular, Universidad Miguel Hernandez, 03202 Elche (Alicante) (Spain); Unidad AECC de Investigacion Traslacional en Cancer, Hospital Universitario Virgen de la Arrixaca, 30120 Murcia (Spain)

2011-06-10

Signaling through the epidermal growth factor receptor (EGFR) is relevant in glioblastoma. We have determined the effects of the EGFR inhibitor AG1478 in glioblastoma cell lines and found that U87 and LN-229 cells were very sensitive to this drug, since their proliferation diminished and underwent a marked G{sub 1} arrest. T98 cells were a little more refractory to growth inhibition and A172 cells did not undergo a G{sub 1} arrest. This G{sub 1} arrest was associated with up-regulation of p27{sup kip1}, whose protein turnover was stabilized. EGFR autophosphorylation was blocked with AG1478 to the same extent in all the cell lines. Other small-molecule EGFR tyrosine kinase inhibitors employed in the clinic, such as gefitinib, erlotinib and lapatinib, were able to abrogate proliferation of glioblastoma cell lines, which underwent a G{sub 1} arrest. However, the EGFR monoclonal antibody, cetuximab had no effect on cell proliferation and consistently, had no effect on cell cycle either. Similarly, cetuximab did not inhibit proliferation of U87 {Delta}EGFR cells or primary glioblastoma cell cultures, whereas small-molecule EGFR inhibitors did. Activity of downstream signaling molecules of EGFR such as Akt and especially ERK1/2 was interrupted with EGFR tyrosine kinase inhibitors, whereas cetuximab treatment could not sustain this blockade over time. Small-molecule EGFR inhibitors were able to prevent phosphorylation of erbB3 and erbB4, whereas cetuximab only hindered EGFR phosphorylation, suggesting that EGFR tyrosine kinase inhibitors may mediate their anti-proliferative effects through other erbB family members. We can conclude that small-molecule EGFR inhibitors may be a therapeutic approach for the treatment of glioblastoma patients.

Anti-inflammatory and anti-angiogenic activities in vitro of eight diterpenes from Daphne genkwa based on hierarchical cluster and principal component analysis.

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Wang, Ling; Lan, Xin-Yi; Ji, Jun; Zhang, Chun-Feng; Li, Fei; Wang, Chong-Zhi; Yuan, Chun-Su

2018-06-01

Rheumatoid arthritis (RA) is one of the most prevalent chronic inflammatory and angiogenic diseases. The aim of this study was to evaluate the anti-inflammatory and anti-angiogenic activities in vitro of eight diterpenoids isolated from Daphne genkwa. LC-MS was used to identify diterpenes isolated from D. genkwa. The anti-inflammatory and anti-angiogenic activities of eight diterpenoids were evaluated on LPS-induced macrophage RAW264.7 cells and TNF-α-stimulated human umbilical vein endothelial cells (HUVECs) using hierarchical cluster analysis (HCA) and principal component analysis (PCA). The eight diterpenes isolated from D. genkwa were identified as yuanhuaphnin, isoyuanhuacine, 12-O-(2'E,4'E-decadienoyl)-4-hydroxyphorbol-13-acetyl, yuanhuagine, isoyuanhuadine, yuanhuadine, yuanhuaoate C and yuanhuacine. All the eight diterpenes significantly down-regulated the excessive secretion of TNF-α, IL-6, IL-1β and NO in LPS-induced RAW264.7 macrophages. However, only 12-O-(2'E,4'E-decadienoyl)-4-hydroxyphorbol-13-acetyl markedly reduced production of VEGF, MMP-3, ICAM and VCAM in TNF-α-stimulated HUVECs. HCA obtained 4 clusters, containing 12-O-(2'E,4'E-decadienoyl)-4-hydroxyphorbol-13-acetyl, isoyuanhuacine, isoyuanhuadine and five other compounds. PCA showed that the ranking of diterpenes sorted by efficacy from highest to lowest was 12-O-(2'E,4'E-decadienoyl)-4-hydroxyphorbol-13-acetyl, yuanhuaphnin, isoyuanhuacine, yuanhuacine, yuanhuaoate C, yuanhuagine, isoyuanhuadine, yuanhuadine. In conclusion, eight diterpenes isolated from D. genkwa showed different levels of activity in LPS-induced RAW264.7 cells and TNF-α-stimulated HUVECs. The comprehensive evaluation of activity by HCA and PCA indicated that of the eight diterpenes, 12-O-(2'E,4'E-decadienoyl)-4-hydroxyphorbol-13-acetyl was the best, and can be developed as a new drug for RA therapy.

MHC class I signaling in T cells leads to tyrosine kinase activity and PLC-gamma 1 phosphorylation

DEFF Research Database (Denmark)

Skov, S; Odum, Niels; Claesson, M H

1995-01-01

phosphorylation and the subsequent calcium response. The early tyrosine kinase activity was found to be dependent on expression of the TCR/CD3 complex and the CD45 molecule on the surface of the T cells. Furthermore, MHC-I cross-linking was shown to tyrosine phosphorylate PLC-gamma 1 (phospholipase C-gamma 1...

Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39

International Nuclear Information System (INIS)

Mai, Sanyue; Qu, Xiuhua; Li, Ping; Ma, Qingjun; Liu, Xuan; Cao, Cheng

2016-01-01

RBM39, also known as splicing factor HCC1.4, acts as a transcriptional coactivator for the steroid nuclear receptors JUN/AP-1, ESR1/ER-α and ESR2/ER-β. RBM39 is involved in the regulation of the transcriptional responses of these steroid nuclear receptors and promotes transcriptional initiation. In this paper, we report that RBM39 interacts with the nonreceptor tyrosine kinase c-Abl. Both the Src homology (SH) 2 and SH3 domains of c-Abl interact with RBM39. The major tyrosine phosphorylation sites on RBM39 that are phosphorylated by c-Abl are Y95 and Y99, as demonstrated by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) and mutational analysis. c-Abl was shown boost the transcriptional coactivation activity of RBM39 for ERα and PRβ in a tyrosine kinase-dependent manner. The results suggest that mammalian c-Abl plays an important role in steroid hormone receptor-mediated transcription by regulating RBM39. - Highlights: • c-Abl interacts with RBM39. • RBM39 is phosphorylated by c-Abl. • c-Abl regulates transcriptional coactivation activity of RBM39 on the ERα and PRβ.

Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39

Energy Technology Data Exchange (ETDEWEB)

Mai, Sanyue [Beijing Institute of Biotechnology, 27 Taiping Rd, Haidian District, Beijing 100850 (China); Qu, Xiuhua [General Navy Hospital of PLA, 6 Fucheng Rd, Haidian District, Beijing 100037 (China); Li, Ping; Ma, Qingjun [Beijing Institute of Biotechnology, 27 Taiping Rd, Haidian District, Beijing 100850 (China); Liu, Xuan, E-mail: liux931932@163.com [Beijing Institute of Biotechnology, 27 Taiping Rd, Haidian District, Beijing 100850 (China); Cao, Cheng, E-mail: cao_c@sohu.com [Beijing Institute of Biotechnology, 27 Taiping Rd, Haidian District, Beijing 100850 (China)

2016-04-22

RBM39, also known as splicing factor HCC1.4, acts as a transcriptional coactivator for the steroid nuclear receptors JUN/AP-1, ESR1/ER-α and ESR2/ER-β. RBM39 is involved in the regulation of the transcriptional responses of these steroid nuclear receptors and promotes transcriptional initiation. In this paper, we report that RBM39 interacts with the nonreceptor tyrosine kinase c-Abl. Both the Src homology (SH) 2 and SH3 domains of c-Abl interact with RBM39. The major tyrosine phosphorylation sites on RBM39 that are phosphorylated by c-Abl are Y95 and Y99, as demonstrated by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) and mutational analysis. c-Abl was shown boost the transcriptional coactivation activity of RBM39 for ERα and PRβ in a tyrosine kinase-dependent manner. The results suggest that mammalian c-Abl plays an important role in steroid hormone receptor-mediated transcription by regulating RBM39. - Highlights: • c-Abl interacts with RBM39. • RBM39 is phosphorylated by c-Abl. • c-Abl regulates transcriptional coactivation activity of RBM39 on the ERα and PRβ.

HER2 mutated breast cancer responds to treatment with single agent neratinib, a second generation HER2/EGFR tyrosine kinase inhibitor

Science.gov (United States)

Ben–Baruch, Noa Efrat; Bose, Ron; Kavuri, Shyam M.; Ma, Cynthia X.; Ellis, Matthew J.

2015-01-01

Activating mutations in the HER2 tyrosine kinase have been identified in human breast cancers that lack HER2 gene amplification. These patients are not candidates for HER2 targeted drugs under current standards of care, but preclinical data strongly suggest that these patients will benefit from anti-HER2 drugs. In this case report, we describe a young woman with metastatic breast cancer whose tumor was found to carry a HER2 L755S mutation, which is in the kinase domain of HER2. Treatment with the second generation HER2/EGFR tyrosine kinase inhibitor, neratinib, resulted in partial response and dramatic improvement in the patient’s function status. This partial response lasted 11 months and when the patient’s cancer progressed, she was treated with neratinib plus capecitabine and her cancer again responded. This second response parallels the benefit seen with continuing trastuzumab in HER2 amplified breast cancer after disease progression. This case is the first report, to our knowledge, of successful single agent treatment of HER2 mutated breast cancer. Two clinical trials of neratinib for HER2 mutated, metastatic breast cancer are currently enrolling patients. Further, data from The Cancer Genome Atlas project have identified HER2 mutations in a wide range of solid tumors, including bladder, colorectal, and non-small cell lung cancer, suggesting that clinical trials of neratinib or neratinib-based combinations for HER2 mutated solid tumors is warranted. PMID:26358790

HER2-Mutated Breast Cancer Responds to Treatment With Single-Agent Neratinib, a Second-Generation HER2/EGFR Tyrosine Kinase Inhibitor.

Science.gov (United States)

Ben-Baruch, Noa Efrat; Bose, Ron; Kavuri, Shyam M; Ma, Cynthia X; Ellis, Matthew J

2015-09-01

Activating mutations in the HER2 tyrosine kinase have been identified in human breast cancers that lack HER2 gene amplification. These patients are not candidates for HER2-targeted drugs under current standards of care, but preclinical data strongly suggest that these patients will benefit from anti-HER2 drugs. This case report describes a young woman with metastatic breast cancer whose tumor was found to carry a HER2 L755S mutation, which is in the kinase domain of HER2. Treatment with the second-generation HER2/EGFR tyrosine kinase inhibitor neratinib resulted in partial response and dramatic improvement in the patient's functional status. This partial response lasted 11 months, and when the patient's cancer progressed, she was treated with neratinib plus capecitabine and her cancer again responded. This second response parallels the benefit seen with continuing trastuzumab in HER2-amplified breast cancer after disease progression. This case represents the first report, to our knowledge, of successful single-agent treatment of HER2-mutated breast cancer. Two clinical trials of neratinib for HER2-mutated metastatic breast cancer are currently enrolling patients. Further, data from The Cancer Genome Atlas project have identified HER2 mutations in a wide range of solid tumors, including bladder, colorectal, and non-small cell lung cancers, suggesting that clinical trials of neratinib or neratinib-based combinations for HER2-mutated solid tumors is warranted. Copyright © 2015 by the National Comprehensive Cancer Network.

rse, a novel receptor-type tyrosine kinase with homology to Axl/Ufo, is expressed at high levels in the brain.

Science.gov (United States)

Mark, M R; Scadden, D T; Wang, Z; Gu, Q; Goddard, A; Godowski, P J

1994-04-08

We have isolated cDNA clones that encode the human and murine forms of a novel receptor-type tyrosine kinase termed Rse. Sequence analysis indicates that human Rse contains 890 amino acids, with an extracellular region composed of two immunoglobulin-like domains followed by two fibronectin type III domains. Murine Rse contains 880 amino acids and shares 90% amino acid identity with its human counterpart. Rse is structurally similar to the receptor-type tyrosine kinase Axl/Ufo, and the two proteins have 35 and 63% sequence identity in their extracellular and intracellular domains, respectively. To study the synthesis and activation of this putative receptor-type tyrosine kinase, we constructed a version of Rse (termed gD-Rse, where gD represents glycoprotein D) that contains an NH2-terminal epitope tag. NIH3T3 cells were engineered to express gD-Rse, which could be detected at the cell surface by fluorescence-activated cell sorting. Moreover, gD-Rse was rapidly phosphorylated on tyrosine residues upon incubation of the cells with an antibody directed against the epitope tag, suggesting that rse encodes an active tyrosine kinase. In the human tissues we examined, the highest level of expression of rse mRNA was observed in the brain; rse mRNA was also detected in the premegakaryocytopoietic cell lines CMK11-5 and Dami. The gene for rse was localized to human chromosome 15.

Domains of the growth hormone receptor required for association and activation of JAK2 tyrosine kinase

DEFF Research Database (Denmark)

VanderKuur, J A; Wang, X; Zhang, L

1994-01-01

Growth hormone (GH) has recently been shown to activate the GH receptor (GHR)-associated tyrosine kinase JAK2. In the present study, regions of the GHR required for JAK2 association with GHR were identified. GH-dependent JAK2 association with GHR was detected in Chinese hamster ovary (CHO) cells...... and RIN-5AH cells, the ability of JAK2 to associate with the mutated GHR was found to correlate with GH-dependent activation of JAK2, tyrosyl phosphorylation of GHR (in the case of GHR1-638 and GHR1-454), and the ability of the GHR to copurify with tyrosine kinase activity. In CHO cells expressing mutated......, and that tyrosines in the N-terminal half of the cytoplasmic domain of the GHR are phosphorylated by JAK2. The finding that a specific interaction with the C-terminal half of GHR appears to be necessary for p97 phosphorylation indicates that while JAK2 activation may be necessary for a full biological response to GH...

Single-label kinase and phosphatase assays for tyrosine phosphorylation using nanosecond time-resolved fluorescence detection.

Science.gov (United States)

Sahoo, Harekrushna; Hennig, Andreas; Florea, Mara; Roth, Doris; Enderle, Thilo; Nau, Werner M

2007-12-26

The collision-induced fluorescence quenching of a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) by hydrogen atom abstraction from the tyrosine residue in peptide substrates was introduced as a single-labeling strategy to assay the activity of tyrosine kinases and phosphatases. The assays were tested for 12 different combinations of Dbo-labeled substrates and with the enzymes p60c-Src Src kinase, EGFR kinase, YOP protein tyrosine phosphatase, as well as acid and alkaline phosphatases, thereby demonstrating a broad application potential. The steady-state fluorescence changed by a factor of up to 7 in the course of the enzymatic reaction, which allowed for a sufficient sensitivity of continuous monitoring in steady-state experiments. The fluorescence lifetimes (and intensities) were found to be rather constant for the phosphotyrosine peptides (ca. 300 ns in aerated water), while those of the unphosphorylated peptides were as short as 40 ns (at pH 7) and 7 ns (at pH 13) as a result of intramolecular quenching. Owing to the exceptionally long fluorescence lifetime of Dbo, the assays were alternatively performed by using nanosecond time-resolved fluorescence (Nano-TRF) detection, which leads to an improved discrimination of background fluorescence and an increased sensitivity. The potential for inhibitor screening was demonstrated through the inhibition of acid and alkaline phosphatases by molybdate.

Tumor suppressor function of Bruton tyrosine kinase is independent of its catalytic activity

NARCIS (Netherlands)

S. Middendorp; A.J.E. Zijlstra (Esther); R. Kersseboom (Rogier); G.M. Dingjan (Gemma); H. Jumaa; R.W. Hendriks (Rudi)

2005-01-01

textabstractDuring B-cell development in the mouse, Bruton tyrosine kinase (Btk) and the adaptor protein SLP-65 (Src homology 2 [SH2] domain-containing leukocyte protein of 65 kDa) limit the expansion and promote the differentiation of pre-B cells. Btk is thought to mainly function

Three Paths to Better Tyrosine Kinase Inhibition Behind the Blood-Brain Barrier in Treating Chronic Myelogenous Leukemia and Glioblastoma with Imatinib

Science.gov (United States)

Kast, Richard E; Focosi, Daniele

2010-01-01

Chronic myelogenous leukemia (CML) can be controlled for years with the tyrosine kinase inhibitor imatinib but because imatinib poorly penetrates the blood-brain barrier (BBB), on occasion, the CML clone will thrive and evolve to an accelerated phase in the resulting imatinib sanctuary within the central nervous system. In this, CML resembles glioblastoma in that imatinib, which otherwise may be effective, cannot get to the tumor. Although a common street drug of abuse, methamphetamine is Food and Drug Administration-approved and marketed as a pharmaceutical drug to treat attention-deficit disorders. It has shown the ability to open the BBB in rodents. We have some clinical hints that it may do so in humans as well. This short note presents three new points potentially leading to better tyrosine kinase inhibition behind the BBB: 1) Pharmaceutical methamphetamine may have a useful role in treating both CML and glioblastoma by allowing higher imatinib concentrations behind the BBB. 2) The old antidepressant and monoamine oxidase inhibitor selegiline, used to treat Parkinson disease, is catabolized to methamphetamine. Selegiline, as a nonscheduled drug,may therefore be an easier way to open the BBB, allowing more effective chemotherapy with tyrosine kinases. 3) Dasatinib is a tyrosine kinase inhibitor with a spectrum of inhibition only partially overlapping that of imatinib and a mechanism of tyrosine kinase inhibition that is different from that of imatinib. The two should be additive. In addition, dasatinib crosses the BBB poorly, and it can therefore be expected to benefit from methamphetamine-assisted entry. PMID:20165690

Combined Targeting of BCL-2 and BCR-ABL Tyrosine Kinase Eradicates Chronic Myeloid Leukemia Stem Cells

Science.gov (United States)

Mak, Po Yee; Mu, Hong; Zhou, Hongsheng; Mak, Duncan H.; Schober, Wendy; Leverson, Joel D.; Zhang, Bin; Bhatia, Ravi; Huang, Xuelin; Cortes, Jorge; Kantarjian, Hagop; Konopleva, Marina

2016-01-01

BCR-ABL tyrosine kinase inhibitors (TKIs) are effective against chronic myeloid leukemia (CML), but they rarely eliminate CML stem cells. Disease relapse is common upon therapy cessation, even in patients with complete molecular responses. Furthermore, once CML progresses to blast crisis (BC), treatment outcomes are dismal. We hypothesized that concomitant targeting of BCL-2 and BCR-ABL tyrosine kinase could overcome these limitations. We demonstrate increased BCL-2 expression at the protein level in bone marrow cells, particularly in Lin−Sca-1+cKit+ cells of inducible CML in mice as determined by CyTOF mass cytometry. Further, selective inhibition of BCL-2, aided by TKI-mediated MCL-1 and BCL-XL inhibition, markedly decreased leukemic Lin−Sca-1+cKit+ cell numbers and long-term stem cell frequency, and prolonged survival in a murine CML model. Additionally, this combination effectively eradicated CD34+CD38−, CD34+CD38+, and quiescent stem/progenitor CD34+ cells from BC CML patient samples. Our results suggest that BCL-2 is a key survival factor for CML stem/progenitor cells and that combined inhibition of BCL-2 and BCR-ABL tyrosine kinase has the potential to significantly improve depth of response and cure rates of chronic phase and BC CML. PMID:27605552

Activation of the protein tyrosine phosphatase SHP2 via the interleukin-6 signal transducing receptor protein gp130 requires tyrosine kinase Jak1 and limits acute-phase protein expression.

Science.gov (United States)

Schaper, F; Gendo, C; Eck, M; Schmitz, J; Grimm, C; Anhuf, D; Kerr, I M; Heinrich, P C

1998-11-01

Stimulation of the interleukin-6 (IL-6) signalling pathway occurs via the IL-6 receptor-glycoprotein 130 (IL-6R-gp130) receptor complex and results in the regulation of acute-phase protein genes in liver cells. Ligand binding to the receptor complex leads to tyrosine phosphorylation and activation of Janus kinases (Jak), phosphorylation of the signal transducing subunit gp130, followed by recruitment and phosphorylation of the signal transducer and activator of transcription factors STAT3 and STAT1 and the src homology domain (SH2)-containing protein tyrosine phosphatase (SHP2). The tyrosine phosphorylated STAT factors dissociate from the receptor, dimerize and translocate to the nucleus where they bind to enhancer sequences of IL-6 target genes. Phosphorylated SHP2 is able to bind growth factor receptor bound protein (grb2) and thus might link the Jak/STAT pathway to the ras/raf/mitogen-activated protein kinase pathway. Here we present data on the dose-dependence, kinetics and kinase requirements for SHP2 phosphorylation after the activation of the signal transducer, gp130, of the IL-6-type family receptor complex. When human fibrosarcoma cell lines deficient in Jak1, Jak2 or tyrosine kinase 2 (Tyk2) were stimulated with IL-6-soluble IL-6R complexes it was found that only in Jak1-, but not in Jak 2- or Tyk2-deficient cells, SHP2 activation was greatly impaired. It is concluded that Jak1 is required for the tyrosine phosphorylation of SHP2. This phosphorylation depends on Tyr-759 in the cytoplasmatic domain of gp130, since a Tyr-759-->Phe exchange abrogates SHP2 activation and in turn leads to elevated and prolonged STAT3 and STAT1 activation as well as enhanced acute-phase protein gene induction. Therefore, SHP2 plays an important role in acute-phase gene regulation.

Tyrosine phosphorylation of the BRI1 receptor kinase occurs via a posttranslational modification and is activated by the juxtamembrane domain

Science.gov (United States)

In metazoans, receptor kinases control many essential processes related to growth and development and response to the environment. The receptor kinases in plants and animals are structurally similar but evolutionarily distinct from one another, and thus while most animal receptor kinases are tyrosin...

Metazoan-like signaling in a unicellular receptor tyrosine kinase

Directory of Open Access Journals (Sweden)

Schultheiss Kira P

2013-02-01

Full Text Available Abstract Background Receptor tyrosine kinases (RTKs are crucial components of signal transduction systems in multicellular animals. Surprisingly, numerous RTKs have been identified in the genomes of unicellular choanoflagellates and other protists. Here, we report the first biochemical study of a unicellular RTK, namely RTKB2 from Monosiga brevicollis. Results We cloned, expressed, and purified the RTKB2 kinase, and showed that it is enzymatically active. The activity of RTKB2 is controlled by autophosphorylation, as in metazoan RTKs. RTKB2 possesses six copies of a unique domain (designated RM2 in its C-terminal tail. An isolated RM2 domain (or a synthetic peptide derived from the RM2 sequence served as a substrate for RTKB2 kinase. When phosphorylated, the RM2 domain bound to the Src homology 2 domain of MbSrc1 from M. brevicollis. NMR structural studies of the RM2 domain indicated that it is disordered in solution. Conclusions Our results are consistent with a model in which RTKB2 activation stimulates receptor autophosphorylation within the RM2 domains. This leads to recruitment of Src-like kinases (and potentially other M. brevicollis proteins and further phosphorylation, which may serve to increase or dampen downstream signals. Thus, crucial features of signal transduction circuitry were established prior to the evolution of metazoans from their unicellular ancestors.

Mechanical stretch endows mesenchymal stem cells stronger angiogenic and anti-apoptotic capacities via NFκB activation

International Nuclear Information System (INIS)

Zhu, Zhuoli; Gan, Xueqi; Fan, Hongyi; Yu, Haiyang

2015-01-01

Mesenchymal stem cells (MSCs) have been broadly used for tissue regeneration and repair due to their broad differentiation potential and potent paracrine properties such as angiogenic capacity. Strategies to increase their survival rate after transplantation and the angiogenic ability are of priority for the utility of MSCs. In this study, we found that mechanical stretch (10% extension, 30 cycles/min cyclic stretch) preconditioning increase the angiogenic capacity via VEGFA induction. In addition, mechanical stretch also increases the survival rate of mesenchymal stem cells under nutrients deprivation. Consistent with the increase VEGFA expression and resistance to apoptosis, nuclear localization of NFκB activity p65 increased upon mechanical stretch. Inhibition of NFκB activity by BAY 11-708 blocks the pro-angiogenesis and anti-apoptosis function of mechanical stretch. Taken together, our findings here raise the possibility that mechanical stretch preconditioning might enhance the therapeutic efficacy of mesenchymal stem cells. - Highlights: • Mechanical stretch increases the angiogenic capacity via VEGFA induction in MSCs. • Mechanical stretch increases the survival rate of MSCs under nutrients deprivation. • Mechanical stretch manipulates MSCs via the activation of NFκB.

Mechanical stretch endows mesenchymal stem cells stronger angiogenic and anti-apoptotic capacities via NFκB activation

Energy Technology Data Exchange (ETDEWEB)

Zhu, Zhuoli; Gan, Xueqi [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Fan, Hongyi [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Department of Applied Mechanics, College of Architecture and Environment, Sichuan University, Chengdu 610065 (China); Yu, Haiyang, E-mail: yhyang6812@foxmail.com [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China)

2015-12-25

Mesenchymal stem cells (MSCs) have been broadly used for tissue regeneration and repair due to their broad differentiation potential and potent paracrine properties such as angiogenic capacity. Strategies to increase their survival rate after transplantation and the angiogenic ability are of priority for the utility of MSCs. In this study, we found that mechanical stretch (10% extension, 30 cycles/min cyclic stretch) preconditioning increase the angiogenic capacity via VEGFA induction. In addition, mechanical stretch also increases the survival rate of mesenchymal stem cells under nutrients deprivation. Consistent with the increase VEGFA expression and resistance to apoptosis, nuclear localization of NFκB activity p65 increased upon mechanical stretch. Inhibition of NFκB activity by BAY 11-708 blocks the pro-angiogenesis and anti-apoptosis function of mechanical stretch. Taken together, our findings here raise the possibility that mechanical stretch preconditioning might enhance the therapeutic efficacy of mesenchymal stem cells. - Highlights: • Mechanical stretch increases the angiogenic capacity via VEGFA induction in MSCs. • Mechanical stretch increases the survival rate of MSCs under nutrients deprivation. • Mechanical stretch manipulates MSCs via the activation of NFκB.

Progranulin and the receptor tyrosine kinase EphA2, partners in crime?

Science.gov (United States)

Chitramuthu, Babykumari; Bateman, Andrew

2016-01-01

Progranulin is a secreted protein with roles in tumorigenesis, inflammation, and neurobiology, but its signaling receptors have remained unclear. In this issue, Neill et al. (2016. J. Cell Biol. https://doi.org/10.1083/jcb.201603079) identify the tyrosine kinase EphA2 as a strong candidate for such a receptor, providing insight into progranulin and EphA2 signaling. PMID:27903608

LONG-TERM RESULTS OF TARGET THERAPY WITH FIRST AND * SECOND-LINE TYROSINE KINASE INHIBITORS IN PATIENTS WITH CHRONIC MYELOID LEUKEMIA

Directory of Open Access Journals (Sweden)

L. L. Vysotskaya

2015-01-01

Full Text Available Aim: To assess long-term efficacy of firstand second-line tyrosine kinase inhibitors in non-selected patients with chronic myeloid leukemia in a real-life clinical setting.Materials and methods: The assessment is based on long-term results of a prospective single center comparative clinical trial that was based on non-selected groups of 116 patients with various stages of chronic myeloid leukemia being treated with a first generation tyrosine kinase inhibitor imatinib, and of 44 patients being treated with a second generation tyrosine kinase inhibitor nilotinib. We analyzed all-cause mortality, progression-free survival from April 2005 to April 2013, with a median of the follow-up of 128 months.Results: In 116 patients with chronic myeloid leukemia treated with imatinib, the Kaplan-Meier survival estimate was 120 months. In 44 patients at an early chronic phase, 5-year overall survival and progression-free survival was 93.2% and 8-year overall and progression-free survival was 79.5%. In 44 patients at a late chronic stage, 5-year overall and progression-free survival was 95.5%, 8-year overall and progression-free survival, 72.7%. In 28 patients at acceleration phase, 5-years overall survival was 78.6% and 8-year overall survival, 46%. Median of overall survival in patients treated with nilotinib was not reached. During 78.6 months of combination treatment with cytotoxic agents, tyrosine kinase inhibitors of the first (imatinib and second line (nilotinib, overall survival was 100%.Conclusion: In clinical practice, inclusion of patients with chronic myeloid leukemia and imatinib resistance (disease relapse or imatinib intolerance into the treatment program with frontline therapy with general cytotoxic agents and thereafter with firstand second-line tyrosine kinase inhibitors significantly improves overall survival.

Phosphorylated human prolactin (S179D-hPRL) is a potent anti-angiogenic hormone in vitro and in vivo

International Nuclear Information System (INIS)

Ueda, Eric Kinnosuke Martins

2006-01-01

previously shown, S179D-hPRL blocked extracellular regulated kinase (ERK) phosphorylation in response to bFGF, but, in addition, continued co-incubation showed a delayed and prolonged activation of ERK. PD98059 [a specific mitogen-activated protein kinase (MAPkinase) inhibitor] inhibited this delayed activation of ERK and the effects of S179D-hPRL on all parameters except p53, or activity of the Bax promoter. We conclude that low doses of S179D-hPRL block bFGF-induced ERK signaling and yet activates ERK in a different time frame to elevate p21, and activate the extrinsic pathway. Longer incubations and higher concentrations, however, additionally activate the intrinsic pathway using an alternate intracellular signal. These findings suggest that circulating levels of phosphorylated hPRL may reduce the progression of cancer and, furthermore, that S179D-hPRL may be a useful anti-angiogenic therapeutic. (author)

Anti-angiogenic effect of curcumin, curcumin ethylenediamine derivative and curcumin ethylenediamine manganese complex

OpenAIRE

SUNTORNSUK, Leena; Koizumi, Keiichi; Saitoh, Yurika; Nakamura, ElianeShizuka; KAMMASUD, Naparat; VAJARAGUPTA, Opa; Saiki, Ikuo

2004-01-01

We investigated the anti-angiogenic effect of curcumin, curcumin ethylenediamine derivative (curcumin ED) and curcumin ethylenediamine manganese complex (curcumin EDMn) through the inhibition of the formation of tube-like structures by human umbilical vascular endothelial cells (HUVEC). Curcumin, curcumin ED, curcumin EDMn did not show cytotoxicity to HUVEC at concentrations equal and lower than 10 μM. At the concentration of 10 μM,curcumin, curcumin ED and curcumin EDMn inhibited the tube fo...

Anti-metastatic effects of viral and non-viral mediated Nk4 delivery to tumours.

Science.gov (United States)

Buhles, Alexandra; Collins, Sara A; van Pijkeren, Jan P; Rajendran, Simon; Miles, Michelle; O'Sullivan, Gerald C; O'Hanlon, Deirdre M; Tangney, Mark

2009-03-09

The most common cause of death of cancer sufferers is through the occurrence of metastases. The metastatic behaviour of tumour cells is regulated by extracellular growth factors such as hepatocyte growth factor (HGF), a ligand for the c-Met receptor tyrosine kinase, and aberrant expression/activation of the c-Met receptor is closely associated with metastatic progression. Nk4 (also known as Interleukin (IL)32b) is a competitive antagonist of the HGF c-Met system and inhibits c-Met signalling and tumour metastasis. Nk4 has an additional anti-angiogenic activity independent of its HGF-antagonist function. Angiogenesis-inhibitory as well as cancer-specific apoptosis inducing effects make the Nk4 sequence an attractive candidate for gene therapy of cancer. This study investigates the inhibition of tumour metastasis by gene therapy mediated production of Nk4 by the primary tumour. Optimal delivery of anti-cancer genes is vital in order to achieve the highest therapeutic responses. Non-viral plasmid delivery methods have the advantage of safety and ease of production, providing immediate transgene expression, albeit short-lived in most tumours. Sustained presence of anti-angiogenic molecules is preferable with anti-angiogenic therapies, and the long-term expression mediated by Adeno-associated Virus (AAV) might represent a more appropriate delivery in this respect. However, the incubation time required by AAV vectors to reach appropriate gene expression levels hampers efficacy in many fast-growing murine tumour models. Here, we describe murine trials assessing the effects of Nk4 on the spontaneously metastatic Lewis Lung Carcinoma (LLC) model when delivered to primary tumour via plasmid lipofection or AAV2 vector. Intratumoural AAV-Nk4 administration produced the highest therapeutic response with significant reduction in both primary tumour growth and incidence of lung metastases. Plasmid-mediated therapy also significantly reduced metastatic growth, but with moderate

Insulin treatment promotes tyrosine phosphorylation of PKR and inhibits polyIC induced PKR threonine phosphorylation.

Science.gov (United States)

Swetha, Medchalmi; Ramaiah, Kolluru V A

2015-11-01

Tyrosine phosphorylation of insulin receptor beta (IRβ) in insulin treated HepG2 cells is inversely correlated to ser(51) phosphorylation in the alpha-subunit of eukaryotic initiation factor 2 (eIF2α) that regulates protein synthesis. Insulin stimulates interaction between IRβ and PKR, double stranded RNA-dependent protein kinase, also known as EIF2AK2, and phosphorylation of tyrosine residues in PKR, as analyzed by immunoprecipitation and pull down assays using anti-IRβ and anti-phosphotyrosine antibodies, recombinant IRβ and immunopurified PKR. Further polyIC or synthetic double stranded RNA-induced threonine phosphorylation or activation of immunopurified and cellular PKR is suppressed in the presence of insulin treated purified IRβ and cell extracts. Acute, but not chronic, insulin treatment enhances tyrosine phosphorylation of IRβ, its interaction with PKR and tyrosine phosphorylation of PKR. In contrast, lipopolysaccharide that stimulates threonine phosphorylation of PKR and eIF2α phosphorylation and AG 1024, an inhibitor of the tyrosine kinase activity of IRβ, reduces PKR association with the receptor, IRβ in HepG2 cells. These findings therefore may suggest that tyrosine phosphorylated PKR plays a role in the regulation of insulin induced protein synthesis and in maintaining insulin sensitivity, whereas, suppression of polyIC-mediated threonine phosphorylation of PKR by insulin compromises its ability to fight against virus infection in host cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Inhibiting Src family tyrosine kinase activity blocks glutamate signalling to ERK1/2 and Akt/PKB but not JNK in cultured striatal neurones.

Science.gov (United States)

Crossthwaite, Andrew J; Valli, Haseeb; Williams, Robert J

2004-03-01

Glutamate receptor activation of mitogen-activated protein (MAP) kinase signalling cascades has been implicated in diverse neuronal functions such as synaptic plasticity, development and excitotoxicity. We have previously shown that Ca2+-influx through NMDA receptors in cultured striatal neurones mediates the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt/protein kinase B (PKB) through a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathway. Exposing neurones to the Src family tyrosine kinase inhibitor PP2, but not the inactive analogue PP3, inhibited NMDA receptor-induced phosphorylation of ERK1/2 and Akt/PKB in a concentration-dependent manner, and reduced cAMP response element-binding protein (CREB) phosphorylation. To establish a link between Src family tyrosine kinase-mediated phosphorylation and PI 3-kinase signalling, affinity precipitation experiments were performed with the SH2 domains of the PI 3-kinase regulatory subunit p85. This revealed a Src-dependent phosphorylation of a focal adhesion kinase (FAK)-p85 complex on glutamate stimulation. Demonstrating that PI3-kinase is not ubiquitously involved in NMDA receptor signal transduction, the PI 3-kinase inhibitors wortmannin and LY294002 did not prevent NMDA receptor Ca2+-dependent phosphorylation of c-Jun N-terminal kinase 1/2 (JNK1/2). Further, inhibiting Src family kinases increased NMDA receptor-dependent JNK1/2 phosphorylation, suggesting that Src family kinase-dependent cascades may physiologically limit signalling to JNK. These results demonstrate that Src family tyrosine kinases and PI3-kinase are pivotal regulators of NMDA receptor signalling to ERK/Akt and JNK in striatal neurones.

Receptor tyrosine kinase structure and function in health and disease

Directory of Open Access Journals (Sweden)

Oleg A. Karpov

2015-09-01

Full Text Available Receptor tyrosine kinases (RTKs are membrane proteins that control the flow of information through signal transduction pathways, impacting on different aspects of cell function. RTKs are characterized by a ligand-binding ectodomain, a single transmembrane α-helix, a cytosolic region comprising juxtamembrane and kinase domains followed by a flexible C-terminal tail. Somatic and germline RTK mutations can induce aberrant signal transduction to give rise to cardiovascular, developmental and oncogenic abnormalities. RTK overexpression occurs in certain cancers, correlating signal strength and disease incidence. Diverse RTK activation and signal transduction mechanisms are employed by cells during commitment to health or disease. Small molecule inhibitors are one means to target RTK function in disease initiation and progression. This review considers RTK structure, activation, and signal transduction and evaluates biological relevance to therapeutics and clinical outcomes.

Itk tyrosine kinase substrate docking is mediated by a nonclassical SH2 domain surface of PLCgamma1.

Science.gov (United States)

Min, Lie; Joseph, Raji E; Fulton, D Bruce; Andreotti, Amy H

2009-12-15

Interleukin-2 tyrosine kinase (Itk) is a Tec family tyrosine kinase that mediates signaling processes after T cell receptor engagement. Activation of Itk requires recruitment to the membrane via its pleckstrin homology domain, phosphorylation of Itk by the Src kinase, Lck, and binding of Itk to the SLP-76/LAT adapter complex. After activation, Itk phosphorylates and activates phospholipase C-gamma1 (PLC-gamma1), leading to production of two second messengers, DAG and IP(3). We have previously shown that phosphorylation of PLC-gamma1 by Itk requires a direct, phosphotyrosine-independent interaction between the Src homology 2 (SH2) domain of PLC-gamma1 and the kinase domain of Itk. We now define this docking interface using a combination of mutagenesis and NMR spectroscopy and show that disruption of the Itk/PLCgamma1 docking interaction attenuates T cell signaling. The binding surface on PLCgamma1 that mediates recognition by Itk highlights a nonclassical binding activity of the well-studied SH2 domain providing further evidence that SH2 domains participate in important signaling interactions beyond recognition of phosphotyrosine.

Design, Synthesis and Evaluation of Ribose-modified Anilinopyrimidine Derivatives as EGFR Tyrosine Kinase Inhibitors

Science.gov (United States)

Hu, Xiuqin; Wang, Disha; Tong, Yi; Tong, Linjiang; Wang, Xia; Zhu, Lili; Xie, Hua; Li, Shiliang; Yang, You; Xu, Yufang

2017-11-01

The synthesis of a series of ribose-modified anilinopyrimidine derivatives was efficiently achieved by utilizing DBU or tBuOLi-promoted coupling of ribosyl alcohols with 2,4,5-trichloropyrimidine as key step. Preliminary biological evaluation of this type of compounds as new EGFR tyrosine kinase inhibitors for combating EGFR L858R/T790M mutant associated with drug resistance in the treatment of non-small cell lung cancer revealed that 3-N-acryloyl-5-O-anilinopyrimidine ribose derivative 1a possessed potent and specific inhibitory activity against EGFR L858R/T790M over WT EGFR. Based upon molecular docking studies of the binding mode between compound 1a and EGFR, the distance between the Michael receptor and the pyrimidine scaffold is considered as an important factor for the inhibitory potency and future design of selective EGFR tyrosine kinase inhibitors against EGFR L858R/T790M mutants.

Crystal Structure of Human Dual-Specificity Tyrosine-Regulated Kinase 3 Reveals New Structural Features and Insights into its Auto-phosphorylation.

Science.gov (United States)

Kim, Kuglae; Cha, Jeong Seok; Cho, Yong-Soon; Kim, Hoyoung; Chang, Nienping; Kim, Hye-Jung; Cho, Hyun-Soo

2018-04-07

Dual-specificity tyrosine-regulated kinases (DYRKs) auto-phosphorylate a critical tyrosine residue in their activation loop and phosphorylate their substrate on serine and threonine residues. The auto-phosphorylation occurs intramolecularly and is a one-off event. DYRK3 is selectively expressed at a high level in hematopoietic cells and attenuates erythroblast development, leading to anemia. In the present study, we determined the crystal structure of the mature form of human DYRK3 in complex with harmine, an ATP competitive inhibitor. The crystal structure revealed a phosphorylation site, residue S350, whose phosphorylation increases the stability of DYRK3 and enhances its kinase activity. In addition, our structural and biochemical assays suggest that the N-terminal auto-phosphorylation accessory domain stabilizes the DYRK3 protein, followed by auto-phosphorylation of the tyrosine of the activation loop, which is important for kinase activity. Finally, our docking analysis provides information for the design of novel and potent therapeutics to treat anemia. Copyright © 2018 Elsevier Ltd. All rights reserved.

A Novel Natural Product-Derived Compound, Vestaine A1, Exerts both Pro-Angiogenic and Anti-Permeability Activity via a Different Pathway from VEGF

Directory of Open Access Journals (Sweden)

Yoko Ishimoto

2016-10-01

Full Text Available Background/Aims: Vascular endothelial growth factor (VEGF is a key molecule in the regulation of both angiogenesis and vascular permeability. However, it is known that overproduction of VEGF induces abnormal blood vessel formation and these vessels cause several disease pathologies, such as diabetic retinopathy. The purpose of this study was to find novel vasoactive compounds which have different properties from VEGF. Methods/Results: We screened a natural product library using a co-culture angiogenic assay of endothelial cells and fibroblasts. By focusing on morphological changes of endothelial cells, we isolated the novel compounds vestaine A1 and vestaine B1 from the cultured broth of an actinomycete strain, Streptomyces sp. SANK 63697. Vestaine A1 enhanced tube formation of endothelial cells in Matrigel and suppressed cell death induced by serum deprivation. Vestaine A1 activated both MEK1/2 and PI-3 kinase pathways independently of the VEGF pathway in a dose- and time-dependent fashion. Finally, vestaine A1 potently suppressed VEGF-induced vascular permeability both in vitro and in vivo. Conclusion: Vestaine A1 has the potential to exhibit both pro-angiogenic and anti-permeability properties, and would therefore be useful for therapeutic treatment for abnormal vascular permeability-related diseases.

Induction of matrix metalloproteinase-2 by tenascin-X deficiency is mediated through the c-Jun N-terminal kinase and protein tyrosine kinase phosphorylation pathway

International Nuclear Information System (INIS)

Matsumoto, Ken-ichi; Minamitani, Takeharu; Orba, Yasuko; Sato, Mami; Sawa, Hirofumi; Ariga, Hiroyoshi

2004-01-01

The results of our previous study showed that tumor invasion and metastasis are promoted in extracellular matrix (ECM) tenascin-X-deficient (TNX-/-) mice via increased expression of matrix metalloproteinases (MMPs). However, little is known about the relationship between TNX deficiency and activation of MMP genes. In this study, we investigated the molecular mechanism by which TNX deficiency activates the MMP-2 gene. We examined the intracellular signaling pathways that regulate gene expression of the proteinase in isolated fibroblasts. Results of gelatin zymography showed that MMP-2 was induced to a greater extent in TNX-/- fibroblasts embedded in type I collagen than in wild-type fibroblasts. RT-PCR analysis revealed that the increased level of MMP-2 expression was caused at the transcription level. Conversely, stable overexpression of TNX in a fibroblast cell line reduced MMP-2 expression and suppressed MMP-2 promoter activity. In addition, treatment of TNX-/- fibroblasts with SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, and genistein, a tyrosine kinase inhibitor, suppressed the increased level of proMMP-2 and increased MMP-2 promoter activity in TNX-/- fibroblasts. Furthermore, increased activation of JNK and tyrosine phosphorylation of certain proteins were observed in TNX-/- fibroblasts. These findings suggest that induction of MMP-2 by TNX deficiency is mediated, at least in part, through the JNK and protein tyrosine kinase phosphorylation pathway

A ternary-complex of a suicide gene, a RAGE-binding peptide, and polyethylenimine as a gene delivery system with anti-tumor and anti-angiogenic dual effects in glioblastoma.

Science.gov (United States)

Choi, Eunji; Oh, Jungju; Lee, Dahee; Lee, Jaewon; Tan, Xiaonan; Kim, Minkyung; Kim, Gyeungyun; Piao, Chunxian; Lee, Minhyung

2018-04-13

The receptor for advanced glycation end-products (RAGE) is involved in tumor angiogenesis. Inhibition of RAGE might be an effective anti-angiogenic therapy for cancer. In this study, a cationic RAGE-binding peptide (RBP) was produced as an antagonist of RAGE, and a ternary-complex consisting of RBP, polyethylenimine (2 kDa, PEI2k), and a suicide gene (pHSVtk) was developed as a gene delivery system with dual functions: the anti-tumor effect of pHSVtk and anti-angiogenic effect of RBP. As an antagonist of RAGE, RBP decreased the secretion of vascular-endothelial growth factor (VEGF) in activated macrophages and reduced the tube-formation of endothelial cells in vitro. In in vitro transfection assays, the RBP/PEI2k/plasmid DNA (pDNA) ternary-complex had higher transfection efficiency than the PEI2k/pDNA binary-complex. In an intracranial glioblastoma animal model, the RBP/PEI2k/pHSVtk ternary-complex reduced α-smooth muscle actin expression, suggesting that the complex has an anti-angiogenic effect. In addition, the ternary-complex had higher pHSVtk delivery efficiency than the PEI2k/pHSVtk and PEI25k/pHSVtk binary-complexes in an animal model. As a result, the ternary-complex induced apoptosis and reduced tumor volume more effectively than the PEI2k/pHSVtk and PEI25k/pHSVtk binary-complexes. In conclusion, due to its dual anti-tumor and anti-angiogenesis effects, the RBP/PEI2k/pHSVtk ternary-complex might be an efficient gene delivery system for the treatment of glioblastoma. Copyright © 2018 Elsevier B.V. All rights reserved.

Activation of Bacillus subtilis Ugd by the BY-Kinase PtkA Proceeds via Phosphorylation of Its Residue Tyrosine 70

DEFF Research Database (Denmark)

Petranovic, Dina; Grangeasse, C.; Macek, B.

2009-01-01

-specific phosphoproteomic study indicated that tyrosine 70 is phosphorylated in the Bacillus subtilis UDP-glucose dehydrogenase Ugd. In this study we confirm that this tyrosine 70 is indeed the main residue phosphorylated by the cognate BY-kinase PtkA. Homology-based modeling of the Ugd structure using structures from UDP...

The Syk protein tyrosine kinase can function independently of CD45 or Lck in T cell antigen receptor signaling

NARCIS (Netherlands)

Chu, D. H.; Spits, H.; Peyron, J. F.; Rowley, R. B.; Bolen, J. B.; Weiss, A.

1996-01-01

The protein tyrosine phosphatase CD45 is a critical component of the T cell antigen receptor (TCR) signaling pathway, acting as a positive regulator of Src family protein tyrosine kinases (PTKs) such as Lck. Most CD45-deficient human and murine T cell lines are unable to signal through their TCRs.

A threshold model for receptor tyrosine kinase signaling specificity and cell fate determination [version 1; referees: 4 approved

Directory of Open Access Journals (Sweden)

Allen Zinkle

2018-06-01

Full Text Available Upon ligand engagement, the single-pass transmembrane receptor tyrosine kinases (RTKs dimerize to transmit qualitatively and quantitatively different intracellular signals that alter the transcriptional landscape and thereby determine the cellular response. The molecular mechanisms underlying these fundamental events are not well understood. Considering recent insights into the structural biology of fibroblast growth factor signaling, we propose a threshold model for RTK signaling specificity in which quantitative differences in the strength/longevity of ligand-induced receptor dimers on the cell surface lead to quantitative differences in the phosphorylation of activation loop (A-loop tyrosines as well as qualitative differences in the phosphorylation of tyrosines mediating substrate recruitment. In this model, quantitative differences on A-loop tyrosine phosphorylation result in gradations in kinase activation, leading to the generation of intracellular signals of varying amplitude/duration. In contrast, qualitative differences in the pattern of tyrosine phosphorylation on the receptor result in the recruitment/activation of distinct substrates/intracellular pathways. Commensurate with both the dynamics of the intracellular signal and the types of intracellular pathways activated, unique transcriptional signatures are established. Our model provides a framework for engineering clinically useful ligands that can tune receptor dimerization stability so as to bias the cellular transcriptome to achieve a desired cellular output.

Angiogenic factors for prediction of preeclampsia and intrauterine growth restriction onset in high-risk women: AngioPred study.

Science.gov (United States)

Raia-Barjat, Tiphaine; Prieux, Carole; Gris, Jean-Christophe; Chapelle, Céline; Laporte, Silvy; Chauleur, Céline

2017-09-22

The study aimed to compare the level of two angiogenic factors, soluble fms-like tyrosine kinase-1 (sFlt1) and soluble endoglin (sEng), for the prediction of preeclampsia and intrauterine growth restriction in high-risk pregnant women. A prospective multicenter cohort study of 200 pregnant patients was conducted between June 2008 and October 2010. sFlt1 and sEng were measured by enzyme-linked immunosorbent assay. Forty-five patients developed a placenta-mediated adverse pregnancy outcome. Plasma levels of sFlt1 and sEng were higher in patients who will experience a preeclampsia at 28, 32, and 36 weeks compared with patients with no complication. The same results were observed for intrauterine growth restriction. Plasma levels of sFlt1 and sEng were not significantly different for patients with preeclampsia compare to patients with intrauterine growth restriction. Patients with early pre-eclampsia (PE) had very high rates of angiogenic factors at 20, 24, and 28 weeks. Patients with late PE and early and late intrauterine growth retardation (IUGR) had high rates at 32 and 36 weeks. In high-risk women, angiogenic factors are disturbed before the onset of preeclampsia and this is true for intrauterine growth restriction.

Activation of phosphatidylinositol-3 kinase by nerve growth factor involves indirect coupling of the trk proto-oncogene with src homology 2 domains.

Science.gov (United States)

Ohmichi, M; Decker, S J; Saltiel, A R

1992-10-01

Growth factor receptor tyrosine kinases can form stable associations with intracellular proteins that contain src homology (SH) 2 domains, including the p85 regulatory subunit of phosphatidylinositol (PI)-3 kinase. The activation of this enzyme by growth factors is evaluated in PC12 pheochromocytoma cells and NIH 3T3 fibroblasts expressing the pp140c-trk nerve growth factor (NGF) receptor (3T3-c-trk). NGF causes the rapid stimulation of PI-3 kinase activity detected in anti-phosphotyrosine, but not in anti-trk, immunoprecipitates. This effect coincides with the tyrosine phosphorylation of two proteins, with molecular masses of of 100 kd and 110 kd, that coimmunoprecipitate with p85. Similar phosphorylation patterns are induced when an immobilized fusion protein containing the amino-terminal SH2 domain of p85 is used to precipitate tyrosine-phosphorylated proteins. Thus, although NGF produces the rapid activation of PI-3 kinase through a mechanism that involves tyrosine phosphorylation, there is no evidence for tyrosine phosphorylation of p85, or for its ligand-dependent association with the NGF receptor. Perhaps another phosphoprotein may link the NGF receptor to this enzyme.

The Receptor Tyrosine Kinase AXL in Cancer Progression

Directory of Open Access Journals (Sweden)

Erinn B. Rankin

2016-11-01

Full Text Available The AXL receptor tyrosine kinase (AXL has emerged as a promising therapeutic target for cancer therapy. Recent studies have revealed a central role of AXL signaling in tumor proliferation, survival, stem cell phenotype, metastasis, and resistance to cancer therapy. Moreover, AXL is expressed within cellular components of the tumor microenvironment where AXL signaling contributes to the immunosuppressive and protumorigenic phenotypes. A variety of AXL inhibitors have been developed and are efficacious in preclinical studies. These agents offer new opportunities for therapeutic intervention in the prevention and treatment of advanced disease. Here we review the literature that has illuminated the cellular and molecular mechanisms by which AXL signaling promotes tumor progression and we will discuss the therapeutic potential of AXL inhibition for cancer therapy.

Nuclear localization of Src-family tyrosine kinases is required for growth factor-induced euchromatinization

International Nuclear Information System (INIS)

Takahashi, Akinori; Obata, Yuuki; Fukumoto, Yasunori; Nakayama, Yuji; Kasahara, Kousuke; Kuga, Takahisa; Higashiyama, Yukihiro; Saito, Takashi; Yokoyama, Kazunari K.; Yamaguchi, Naoto

2009-01-01

Src-family kinases (SFKs), which participate in various signaling events, are found at not only the plasma membrane but also several subcellular compartments, including the nucleus. Nuclear structural changes are frequently observed during transcription, cell differentiation, senescence, tumorigenesis, and cell cycle. However, little is known about signal transduction in the alteration of chromatin texture. Here, we develop a pixel imaging method for quantitatively evaluating chromatin structural changes. Growth factor stimulation increases euchromatic hypocondensation and concomitant heterochromatic hypercondensation in G 1 phase, and the levels reach a plateau by 30 min, sustain for at least 5 h and return to the basal levels after 24 h. Serum-activated SFKs in the nucleus were more frequently detected in the euchromatin areas than the heterochromatin areas. Nuclear expression of kinase-active SFKs, but not unrelated Syk kinase, drastically increases both euchromatinization and heterochromatinization in a manner dependent on the levels of nuclear tyrosine phosphorylation. However, growth factor stimulation does not induce chromatin structural changes in SYF cells lacking SFKs, and reintroduction of one SFK member into SYF cells can, albeit insufficiently, induce chromatin structural changes. These results suggest that nuclear tyrosine phosphorylation by SFKs plays an important role in chromatin structural changes upon growth factor stimulation.

ESCMID Study Group for Infections in Compromised Hosts (ESGICH) Consensus Document on the safety of targeted and biological therapies: an infectious diseases perspective (Intracellular signaling pathways: tyrosine kinase and mTOR inhibitors).

Science.gov (United States)

Reinwald, M; Silva, J T; Mueller, N J; Fortún, J; Garzoni, C; de Fijter, J W; Fernández-Ruiz, M; Grossi, P; Aguado, J M

2018-06-01

The present review is part of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Study Group for Infections in Compromised Hosts (ESGICH) Consensus Document on the safety of targeted and biologic therapies. To review, from an infectious diseases perspective, the safety profile of therapies targeting different intracellular signaling pathways and to suggest preventive recommendations. Computer-based Medline searches with MeSH terms pertaining to each agent or therapeutic family. Although BCR-ABL tyrosine kinase inhibitors modestly increase the overall risk of infection, dasatinib has been associated with cytomegalovirus and hepatitis B virus reactivation. BRAF/MEK kinase inhibitors do not significantly affect infection susceptibility. The effect of Bruton tyrosine kinase inhibitors (ibrutinib) among patients with B-cell malignancies is difficult to distinguish from that of previous immunosuppression. However, cases of Pneumocystis jirovecii pneumonia (PCP), invasive fungal infection and progressive multifocal leukoencephalopathy have been occasionally reported. Because phosphatidylinositol-3-kinase inhibitors (idelalisib) may predispose to opportunistic infections, anti-Pneumocystis prophylaxis and prevention strategies for cytomegalovirus are recommended. No increased rates of infection have been observed with venetoclax (antiapoptotic protein Bcl-2 inhibitor). Therapy with Janus kinase inhibitors markedly increases the incidence of infection. Pretreatment screening for chronic hepatitis B virus and latent tuberculosis infection must be performed, and anti-Pneumocystis prophylaxis should be considered for patients with additional risk factors. Cancer patients receiving mTOR inhibitors face an increased incidence of overall infection, especially those with additional risk factors (prior therapies or delayed wound healing). Specific preventive approaches are warranted in view of the increased risk of infection associated with some of the

Discovery of novel EGFR tyrosine kinase inhibitors by structure-based virtual screening.

Science.gov (United States)

Li, Siyuan; Sun, Xianqiang; Zhao, Hongli; Tang, Yun; Lan, Minbo

2012-06-15

By using of structure-based virtual screening, 13 novel epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors were discovered from 197,116 compounds in the SPECS database here. Among them, 8 compounds significantly inhibited EGFR kinase activity with IC(50) values lower than 10 μM. 3-{[1-(3-Chloro-4-fluorophenyl)-3,5-dioxo-4-pyrazolidinylidene]methyl}phenyl 2-thiophenecarboxylate (13), particularly, was the most potent inhibitor possessing the IC(50) value of 3.5 μM. The docking studies also provide some useful information that the docking models of the 13 compounds are beneficial to find a new path for designing novel EGFR inhibitors. Copyright © 2012 Elsevier Ltd. All rights reserved.

The selectivity of receptor tyrosine kinase signaling is controlled by a secondary SH2 domain binding site.

Science.gov (United States)

Bae, Jae Hyun; Lew, Erin Denise; Yuzawa, Satoru; Tomé, Francisco; Lax, Irit; Schlessinger, Joseph

2009-08-07

SH2 domain-mediated interactions represent a crucial step in transmembrane signaling by receptor tyrosine kinases. SH2 domains recognize phosphotyrosine (pY) in the context of particular sequence motifs in receptor phosphorylation sites. However, the modest binding affinity of SH2 domains to pY containing peptides may not account for and likely represents an oversimplified mechanism for regulation of selectivity of signaling pathways in living cells. Here we describe the crystal structure of the activated tyrosine kinase domain of FGFR1 in complex with a phospholipase Cgamma fragment. The structural and biochemical data and experiments with cultured cells show that the selectivity of phospholipase Cgamma binding and signaling via activated FGFR1 are determined by interactions between a secondary binding site on an SH2 domain and a region in FGFR1 kinase domain in a phosphorylation independent manner. These experiments reveal a mechanism for how SH2 domain selectivity is regulated in vivo to mediate a specific cellular process.

Intracellular signaling of the Ufo/Axl receptor tyrosine kinase is mediated mainly by a multi-substrate docking-site.

Science.gov (United States)

Braunger, J; Schleithoff, L; Schulz, A S; Kessler, H; Lammers, R; Ullrich, A; Bartram, C R; Janssen, J W

1997-06-05

Ufo/Axl belongs to a new family of receptor tyrosine kinases with an extracellular structure similar to that of neural cell adhesion molecules. In order to elucidate intracellular signaling, the cytoplasmic moiety of Ufo/Axl was used to screen an expression library according to the CORT (cloning of receptor targets) method. Three putative Ufo substrates were identified: phospholipase Cgamma1 (PLCgamma), as well as p85alpha and p85beta subunits of phosphatidylinositol 3'-kinase (PI3-kinase). Subsequently, chimeric EGFR/Ufo receptors consisting of the extracellular domains of the epidermal growth factor receptor (EGFR) and the transmembrane and intracellular moiety of Ufo were engineered. Using different far-Western blot analyses and coimmunoprecipitation assays, receptor binding of PLCgamma and p85 proteins as well as GRB2, c-src and lck was examined in vitro and in vivo. Competitive inhibition of substrate binding and mutagenesis experiments with EGFR/Ufo constructs revealed C-terminal tyrosine 821 (EILpYVNMDEG) as a docking site for multiple effectors, namely PLCgamma, p85 proteins, GRB2, c-src and lck. Tyrosine 779 (DGLpYALMSRC) demonstrated an additional, but lower binding affinity for the p85 proteins in vitro. In addition, binding of PLCgamma occurred through tyrosine 866 (AGRpYVLCPST). Moreover, our in vivo data indicate that further direct or indirect binding sites for PLCgamma, GRB2, c-src and lck on the human Ufo receptor may exist.

Investigation of the expression of the EphB4 receptor tyrosine kinase in prostate carcinoma

International Nuclear Information System (INIS)

Lee, Yen-Ching; Perren, Janeanne R; Douglas, Evelyn L; Raynor, Michael P; Bartley, Maria A; Bardy, Peter G; Stephenson, Sally-Anne

2005-01-01

The EphB4 receptor tyrosine kinase has been reported as increased in tumours originating from several different tissues and its expression in a prostate cancer xenograft model has been reported. RT-PCR, western blotting and immunohistochemical techniques were used to examine EphB4 expression and protein levels in human prostate cancer cell lines LNCaP, DU145 and PC3. Immunohistochemistry was also used to examine localisation of EphB4 in tissue samples from 15 patients with prostate carcinomas. All three prostate cancer cell lines expressed the EphB4 gene and protein. EphB4 immunoreactivity in vivo was significantly greater in human prostate cancers as compared with matched normal prostate epithelium and there appeared to be a trend towards increased expression with higher grade disease. EphB4 is expressed in prostate cancer cell lines with increased expression in human prostate cancers when compared with matched normal tissue. EphB4 may therefore be a useful anti-prostate cancer target

Icotinib, a selective EGF receptor tyrosine kinase inhibitor, for the treatment of non-small-cell lung cancer.

Science.gov (United States)

Tan, Fenlai; Shi, Yuankai; Wang, Yinxiang; Ding, Lieming; Yuan, Xiaobin; Sun, Yan

2015-01-01

Advanced non-small-cell lung cancer (NSCLC) is the main cause for cancer-related mortality. Treatments for advanced NSCLC are largely palliative and a benefit plateau appears to have reached with the platinum-based chemotherapy regimens. EGF receptor (EGFR) tyrosine kinase inhibitors gefitinib, erlotinib and afatinib came up with prolonged progression-free survival and improved quality of life, especially in EGFR-mutated patients. Icotinib is an oral selective EGFR tyrosine kinase, which was approved by China Food and Drug administration in June 2011 for treating advanced NSCLC. Its approval was based on the registered Phase III trial (ICOGEN), which showed icotinib is noninferior to gefitinib. This review will discuss the role of icotinib in NSCLC, and its potential application and ongoing investigations.

Angiogenic imbalance and diminished matrix metalloproteinase-2 and -9 underlie regional decreases in uteroplacental vascularization and feto-placental growth in hypertensive pregnancy.

Science.gov (United States)

Dias-Junior, Carlos A; Chen, Juanjuan; Cui, Ning; Chiang, Charles L; Zhu, Minglin; Ren, Zongli; Possomato-Vieira, Jose S; Khalil, Raouf A

2017-12-15

Preeclampsia is a form of hypertension-in-pregnancy (HTN-Preg) with unclear mechanism. Generalized reduction of uterine perfusion pressure (RUPP) could be an initiating event leading to uteroplacental ischemia, angiogenic imbalance, and HTN-Preg. Additional regional differences in uteroplacental blood flow could further affect the pregnancy outcome and increase the risk of preeclampsia in twin or multiple pregnancy, but the mechanisms involved are unclear. To test the hypothesis that regional differences in angiogenic balance and matrix metalloproteinases (MMPs) underlie regional uteroplacental vascularization and feto-placental development, we compared fetal and placental growth, and placental and myoendometrial vascularization in the proximal, middle and distal regions of the uterus (in relation to the iliac bifurcation) in normal pregnant (Preg) and RUPP rats. Maternal blood pressure and plasma anti-angiogenic soluble fms-like tyrosine kinase-1 (sFlt-1)/placenta growth factor (PIGF) ratio were higher, and average placentae number, placenta weight, litter size, and pup weight were less in RUPP than Preg rats. The placenta and pup number and weight were reduced, while the number and diameter of placental and adjacent myoendometrial arteries, and MMP-2 and MMP-9 levels/activity were increased, and sFlt-1/PlGF ratio was decreased in distal vs proximal uterus of Preg rats. In RUPP rats, the placenta and pup number and weight, the number and diameter of placental and myoendometrial arteries, and MMP-2 and -9 levels/activity were decreased, and sFlt-1/PlGF ratio was increased in distal vs proximal uterus. Treatment with sFlt-1 or RUPP placenta extract decreased MMP-2 and MMP-9 in distal segments of Preg uterus, and treatment with PIGF or Preg placenta extract restored MMP levels in distal segments of RUPP uterus. Thus, in addition to the general reduction in placental and fetal growth during uteroplacental ischemia, localized angiogenic imbalance and diminished MMP-2

Zinc-chelation contributes to the anti-angiogenic effect of ellagic acid on inhibiting MMP-2 activity, cell migration and tube formation.

Directory of Open Access Journals (Sweden)

Sheng-Teng Huang

Full Text Available BACKGROUND: Ellagic acid (EA, a dietary polyphenolic compound, has been demonstrated to exert anti-angiogenic effect but the detailed mechanism is not yet fully understood. The aim of this study was to investigate whether the zinc chelating activity of EA contributed to its anti-angiogenic effect. METHODS AND PRINCIPAL FINDINGS: The matrix metalloproteinases-2 (MMP-2 activity, a zinc-required reaction, was directly inhibited by EA as examined by gelatin zymography, which was reversed dose-dependently by adding zinc chloride. In addition, EA was demonstrated to inhibit the secretion of MMP-2 from human umbilical vein endothelial cells (HUVECs as analyzed by Western blot method, which was also reversed by the addition of zinc chloride. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK, known to down-regulate the MMP-2 activity, was induced by EA at both the mRNA and protein levels which was correlated well with the inhibition of MMP-2 activity. Interestingly, zinc chloride could also abolish the increase of EA-induced RECK expression. The anti-angiogenic effect of EA was further confirmed to inhibit matrix-induced tube formation of endothelial cells. The migration of endothelial cells as analyzed by transwell filter assay was suppressed markedly by EA dose-dependently as well. Zinc chloride could reverse these two effects of EA also in a dose-dependent manner. Since magnesium chloride or calcium chloride could not reverse the inhibitory effect of EA, zinc was found to be involved in tube formation and migration of vascular endothelial cells. CONCLUSIONS/SIGNIFICANCE: Together these results demonstrated that the zinc chelation of EA is involved in its anti-angiogenic effects by inhibiting MMP-2 activity, tube formation and cell migration of vascular endothelial cells. The role of zinc was confirmed to be important in the process of angiogenesis.

Receptor Tyrosine Kinases as Targets for Treatment of Peripheral Nerve Sheath Tumors in NF 1 Patients

National Research Council Canada - National Science Library

Mautner, Victor-Felix

2007-01-01

.... The only available but unsatisfying therapy is surgical tumor resection. The purpose of this study is the preclinical testing of multiple available tyrosine kinase inhibitors for NF1-associated MPNST using in vitro and in vivo systems...

Combined therapeutic potential of nuclear receptors with receptor tyrosine kinase inhibitors in lung cancer

International Nuclear Information System (INIS)

Wairagu, Peninah M.; Park, Kwang Hwa; Kim, Jihye; Choi, Jong-Whan; Kim, Hyun-Won; Yeh, Byung-Il; Jung, Soon-Hee; Yong, Suk-Joong; Jeong, Yangsik

2014-01-01

Highlights: • The 48 NR genes and 48 biological anti-cancer targets are profiled in paired-cells. • Growth inhibition by NR ligands or TKIs is target receptor level-dependent. • T0901317 with gefitinib/PHA665752 shows additive growth inhibition in lung cells. - Abstract: Cancer heterogeneity is a big hurdle in achieving complete cancer treatment, which has led to the emergence of combinational therapy. In this study, we investigated the potential use of nuclear receptor (NR) ligands for combinational therapy with other anti-cancer drugs. We first profiled all 48 NRs and 48 biological anti-cancer targets in four pairs of lung cell lines, where each pair was obtained from the same patient. Two sets of cell lines were normal and the corresponding tumor cell lines while the other two sets consisted of primary versus metastatic tumor cell lines. Analysis of the expression profile revealed 11 NRs and 15 cancer targets from the two pairs of normal versus tumor cell lines, and 9 NRs and 9 cancer targets from the primary versus metastatic tumor cell lines had distinct expression patterns in each category. Finally, the evaluation of nuclear receptor ligand T0901317 for liver X receptor (LXR) demonstrated its combined therapeutic potential with tyrosine kinase inhibitors. The combined treatment of cMET inhibitor PHA665752 or EGFR inhibitor gefitinib with T0901317 showed additive growth inhibition in both H2073 and H1993 cells. Mechanistically, the combined treatment suppressed cell cycle progression by inhibiting cyclinD1 and cyclinB expression. Taken together, this study provides insight into the potential use of NR ligands in combined therapeutics with other biological anti-cancer drugs

The significance of major and stable molecular responses in chronic myeloid leukemia in the tyrosine kinase inhibitor era

Directory of Open Access Journals (Sweden)

Ilana Zalcberg Renault

2011-12-01

Full Text Available Tyrosine kinase inhibitors have changed the management and outcomes of chronic myeloid leukemia patients. Quantitative polymerase chain reaction is used to monitor molecular responses to tyrosine kinase inhibitors. Molecular monitoring represents the most sensitive tool to judge chronic myeloid leukemia disease course and allows early detection of relapse. Evidence of achieving molecular response is important for several reasons: 1. early molecular response is associated with major molecular response rates at 18-24 months; 2. patients achieving major molecular response are less likely to lose their complete cytogenetic response; 3. a durable, stable major molecular response is associated with increased progression-free survival. However, standardization of molecular techniques is still challenging.

The significance of major and stable molecular responses in chronic myeloid leukemia in the tyrosine kinase inhibitor era

Science.gov (United States)

Renault, Ilana Zalcberg; Scholl, Vanesa; Hassan, Rocio; Capelleti, Paola; de Lima, Marcos; Cortes, Jorge

2011-01-01

Tyrosine kinase inhibitors have changed the management and outcomes of chronic myeloid leukemia patients. Quantitative polymerase chain reaction is used to monitor molecular responses to tyrosine kinase inhibitors. Molecular monitoring represents the most sensitive tool to judge chronic myeloid leukemia disease course and allows early detection of relapse. Evidence of achieving molecular response is important for several reasons: 1. early molecular response is associated with major molecular response rates at 18-24 months; 2. patients achieving major molecular response are less likely to lose their complete cytogenetic response; 3. a durable, stable major molecular response is associated with increased progression-free survival. However, standardization of molecular techniques is still challenging. PMID:23049363

Multi-tyrosine kinase inhibitors in preclinical studies for pediatric CNS AT/RT: Evidence for synergy with Topoisomerase-I inhibition

Directory of Open Access Journals (Sweden)

Jayanthan Aarthi

2011-12-01

Full Text Available Abstract Background Currently, Atypical Teratoid Rhabdoid Tumor (AT/RT constitutes one of the most difficult to treat malignancies in pediatrics. Hence, new knowledge of potential targets for therapeutics and the development of novel treatment approaches are urgently needed. We have evaluated the presence of cytokine pathways and the effects of two clinically available multi-tyrosine kinase inhibitors for cytotoxicity, target modulation and drug combinability against AT/RT cell lines. Results AT/RT cell lines expressed measurable quantities of VEGF, FGF, PDGF and SDF-1, although the absolute amounts varied between the cell lines. The targeted receptor tyrosine kinase inhibitor sorafenib inhibited the key signaling molecule Erk, which was activated following the addition of own conditioned media, suggesting the existence of autocrine/paracrine growth stimulatory pathways. The multi-tyrosine kinase inhibitors sorafenib and sunitinib also showed significant growth inhibition of AT/RT cells and their activity was enhanced by combination with the topoisomerase inhibitor, irinotecan. The loss of cytoplasmic NF-kappa-B in response to irinotecan was diminished by sorafenib, providing evidence for a possible benefit for this drug combination. Conclusions In addition to previously described involvement of insulin like growth factor (IGF family of cytokines, a multitude of other growth factors may contribute to the growth and survival of AT/RT cells. However, consistent with the heterogeneous nature of this tumor, quantitative and qualitative differences may exist among different tumor samples. Multi-tyrosine kinase inhibitors appear to have effective antitumor activity against all cell lines studied. In addition, the target modulation studies and drug combinability data provide the groundwork for additional studies and support the evaluation of these agents in future treatment protocols.

Greater Sensitivity of Blood Pressure Than Renal Toxicity to Tyrosine Kinase Receptor Inhibition With Sunitinib

DEFF Research Database (Denmark)

Lankhorst, Stephanie; Baelde, Hans J; Kappers, Mariëtte H W

2015-01-01

Hypertension and renal injury are off-target effects of sunitinib, a tyrosine kinase receptor inhibitor used for the treatment of various tumor types. Importantly, these untoward effects are accompanied by activation of the endothelin system. Here, we set up a study to explore the dose dependency...

Raman Microspectroscopic Evidence for the Metabolism of a Tyrosine Kinase Inhibitor, Neratinib, in Cancer Cells.

Science.gov (United States)

Aljakouch, Karim; Lechtonen, Tatjana; Yosef, Hesham K; Hammoud, Mohamad K; Alsaidi, Wissam; Kötting, Carsten; Mügge, Carolin; Kourist, Robert; El-Mashtoly, Samir F; Gerwert, Klaus

2018-06-11

Tyrosine kinase receptors are one of the main targets in cancer therapy. They play an essential role in the modulation of growth factor signaling and thereby inducing cell proliferation and growth. Tyrosine kinase inhibitors such as neratinib bind to EGFR and HER2 receptors and exhibit antitumor activity. However, little is known about their detailed cellular uptake and metabolism. Here, we report for the first time the intracellular spatial distribution and metabolism of neratinib in different cancer cells using label-free Raman imaging. Two new neratinib metabolites were detected and fluorescence imaging of the same cells indicate that neratinib accumulates in lysosomes. The results also suggest that both EGFR and HER2 follow the classical endosome lysosomal pathway for degradation. A combination of Raman microscopy, DFT calculations, and LC-MS was used to identify the chemical structure of neratinib metabolites. These results show the potential of Raman microscopy to study drug pharmacokinetics. © 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Alteration of radiation response by two tyrosine kinase inhibitors: STI571 (Glivec) and BIBW 2992

International Nuclear Information System (INIS)

Huguet, F.

2010-01-01

Concurrent chemo-radiation is one of the main weapon in the treatment of cancer. The targeted therapies may act on the mechanisms of tumor resistance to radiation and are therefore very promising in combination with radiotherapy. The STI571 (imatinib or Gleevec) inhibits specifically the Bcr-Abl tyrosine kinase. It leads to radiosensitization in K562 chronic myeloid leukemia cell line by alterations of the cell cycle. The BIBW2992 is a selective inhibitor of EGFR and HER2. The BIBW 2992 shows cytotoxic and radiosensitizing effects on pancreatic adenocarcinoma cells BxPC3 and Capan-2, regardless of KRAS status. The mechanism underlying this radiosensitization is not unequivocal, involving both changes in the cell cycle and induction of mitotic death. Our results show that the combination of an inhibitor of tyrosine kinase with ionizing radiation may lead to a radiosensitization in vitro with mechanisms depending on the type of cell line. (author)

A cross-talk between TrkB and Ret tyrosine kinases receptors mediates neuroblastoma cells differentiation.

Directory of Open Access Journals (Sweden)

Carla Lucia Esposito

Full Text Available Understanding the interplay between intracellular signals initiated by multiple receptor tyrosine kinases (RTKs to give the final cell phenotype is a major pharmacological challenge. Retinoic acid (RA-treatment of neuroblastoma (NB cells implicates activation of Ret and TrkB RTKs as critical step to induce cell differentiation. By studying the signaling interplay between TrkB and Ret as paradigmatic example, here we demonstrate the existence of a cross-talk mechanism between the two unrelated receptors that is needed to induce the cell differentiation. Indeed, we show that TrkB receptor promotes Ret phosphorylation by a mechanism that does not require GDNF. This reveals to be a key mechanism, since blocking either TrkB or Ret by small interfering RNA causes a failure in NB biochemical and morphological differentiation. Our results provide the first evidence that a functional transactivation between distinct tyrosine kinases receptors is required for an important physiological process.

Targeting Bruton Tyrosine Kinase: A novel strategy in the treatment of B-cell lymphomas

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Sklavenitis-Pistofidis R.

2017-06-01

Full Text Available In normal B-cells, Bruton tyrosine kinase (Btk, a non-receptor tyrosine kinase involved in B-cell receptor (BCR signalling, is essential for cell survival and maturation. Not surprisingly, Btk is also implicated in the pathogenesis of B-cell lymphomas, like Chronic Lymphocytic Leukaemia/Small Lymphocytic Lymphoma (CLL/SLL, Mantle Cell Lymphoma (MCL and Waldenström’s Macroglobulinemia (WM, which are driven by aberrant BCR signalling. Thus, targeting Btk represents a promising therapeutic strategy in the treatment of B-cell lymphoma patients. Ibrutinib, a selective Btk inhibitor, has already been approved as second-line treatment of CLL/SLL, MCL and WM patients, while more clinical studies of ibrutinib and novel Btk inhibitors are currently under way. In light of results of the RESONATE-2 trial, the approval of ibrutinib as a first-line treatment of CLL/SLL may well be approaching. Herein, we review Btk’s role in normal and malignant BCR signalling, as well as ibrutinib’s performance in B-cell lymphoma treatment and prognosis.

mTORC2 promotes type I insulin-like growth factor receptor and insulin receptor activation through the tyrosine kinase activity of mTOR.

Science.gov (United States)

Yin, Yancun; Hua, Hui; Li, Minjing; Liu, Shu; Kong, Qingbin; Shao, Ting; Wang, Jiao; Luo, Yuanming; Wang, Qian; Luo, Ting; Jiang, Yangfu

2016-01-01

Mammalian target of rapamycin (mTOR) is a core component of raptor-mTOR (mTORC1) and rictor-mTOR (mTORC2) complexes that control diverse cellular processes. Both mTORC1 and mTORC2 regulate several elements downstream of type I insulin-like growth factor receptor (IGF-IR) and insulin receptor (InsR). However, it is unknown whether and how mTOR regulates IGF-IR and InsR themselves. Here we show that mTOR possesses unexpected tyrosine kinase activity and activates IGF-IR/InsR. Rapamycin induces the tyrosine phosphorylation and activation of IGF-IR/InsR, which is largely dependent on rictor and mTOR. Moreover, mTORC2 promotes ligand-induced activation of IGF-IR/InsR. IGF- and insulin-induced IGF-IR/InsR phosphorylation is significantly compromised in rictor-null cells. Insulin receptor substrate (IRS) directly interacts with SIN1 thereby recruiting mTORC2 to IGF-IR/InsR and promoting rapamycin- or ligand-induced phosphorylation of IGF-IR/InsR. mTOR exhibits tyrosine kinase activity towards the general tyrosine kinase substrate poly(Glu-Tyr) and IGF-IR/InsR. Both recombinant mTOR and immunoprecipitated mTORC2 phosphorylate IGF-IR and InsR on Tyr1131/1136 and Tyr1146/1151, respectively. These effects are independent of the intrinsic kinase activity of IGF-IR/InsR, as determined by assays on kinase-dead IGF-IR/InsR mutants. While both rictor and mTOR immunoprecitates from rictor(+/+) MCF-10A cells exhibit tyrosine kinase activity towards IGF-IR and InsR, mTOR immunoprecipitates from rictor(-/-) MCF-10A cells do not induce IGF-IR and InsR phosphorylation. Phosphorylation-deficient mutation of residue Tyr1131 in IGF-IR or Tyr1146 in InsR abrogates the activation of IGF-IR/InsR by mTOR. Finally, overexpression of rictor promotes IGF-induced cell proliferation. Our work identifies mTOR as a dual-specificity kinase and clarifies how mTORC2 promotes IGF-IR/InsR activation.

Inhibition of Src kinase activity attenuates amyloid associated microgliosis in a murine model of Alzheimer’s disease

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Dhawan Gunjan

2012-07-01

Full Text Available Abstract Background Microglial activation is an important histologic characteristic of the pathology of Alzheimer’s disease (AD. One hypothesis is that amyloid beta (Aβ peptide serves as a specific stimulus for tyrosine kinase-based microglial activation leading to pro-inflammatory changes that contribute to disease. Therefore, inhibiting Aβ stimulation of microglia may prove to be an important therapeutic strategy for AD. Methods Primary murine microglia cultures and the murine microglia cell line, BV2, were used for stimulation with fibrillar Aβ1-42. The non-receptor tyrosine kinase inhibitor, dasatinib, was used to treat the cells to determine whether Src family kinase activity was required for the Aβ stimulated signaling response and subsequent increase in TNFα secretion using Western blot analysis and enzyme-linked immunosorbent assay (ELISA, respectively. A histologic longitudinal analysis was performed using an AD transgenic mouse model, APP/PS1, to determine an age at which microglial protein tyrosine kinase levels increased in order to administer dasatinib via mini osmotic pump diffusion. Effects of dasatinib administration on microglial and astroglial activation, protein phosphotyrosine levels, active Src kinase levels, Aβ plaque deposition, and spatial working memory were assessed via immunohistochemistry, Western blot, and T maze analysis. Results Aβ fibrils stimulated primary murine microglia via a tyrosine kinase pathway involving Src kinase that was attenuated by dasatinib. Dasatinib administration to APP/PS1 mice decreased protein phosphotyrosine, active Src, reactive microglia, and TNFα levels in the hippocampus and temporal cortex. The drug had no effect on GFAP levels, Aβ plaque load, or the related tyrosine kinase, Lyn. These anti-inflammatory changes correlated with improved performance on the T maze test in dasatinib infused animals compared to control animals. Conclusions These data suggest that amyloid

Prevention of pulmonary vascular and myocardial remodeling by the combined tyrosine and serine-/threonine kinase inhibitor, sorafenib, in pulmonary hypertension and right heart failure

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M. Klein

2008-06-01

Full Text Available Inhibition of tyrosine kinases can reverse pulmonary hypertension but little is known about the role of serine-/threonine kinases in vascular and myocardial remodeling. We investigated the effects of sorafenib, an inhibitor of the tyrosine kinases VEGFR, PDGFR and c-kit as well as the serine-/threonine kinase Raf-1, in pulmonary hypertension and right ventricular (RV pressure overload. In monocrotaline treated rats, sorafenib (10 mg·kg–1·d–1 p.o. reduced pulmonary arterial pressure, pulmonary artery muscularization and RV hypertrophy, and improved systemic hemodynamics (table 1. Sorafenib prevented phosphorylation of Raf-1 and suppressed activation of downstream signaling pathways (Erk 1/2. After pulmonary banding, sorafenib, but not the PDGFR/c-KIT/ABL-inhibitor imatinib reduced RV mass and RV filling pressure significantly. Congruent with these results, sorafenib only prevented ERK phosphorylation and vasopressin induced hypertrophy of the cardiomyocyte cell line H9c2 dose dependently (IC50 = 300 nM. Combined inhibition of tyrosine and serine-/threonine kinases by sorafenib prevents vascular and cardiac remodeling in pulmonary hypertension, which is partly mediated via inhibition of the Raf kinase pathway.

Growth of MCF-7 breast cancer cells and efficacy of anti-angiogenic agents in a hydroxyethyl chitosan/glycidyl methacrylate hydrogel.

Science.gov (United States)

Wang, Hejing; Qian, Junmin; Zhang, Yaping; Xu, Weijun; Xiao, Juxiang; Suo, Aili

2017-01-01

Breast cancer negatively affects women's health worldwide. The tumour microenvironment plays a critical role in tumour initiation, proliferation, and metastasis. Cancer cells are traditionally grown in two-dimensional (2D) cultures as monolayers on a flat solid surface lacking cell-cell and cell-matrix interactions. These experimental conditions deviate from the clinical situation. Improved experimental systems that can mimic the in vivo situation are required to discover new therapies, particularly for anti-angiogenic agents that mainly target intercellular factors and play an essential role in treating some cancers. Chitosan can be modified to construct three-dimensional (3D) tumour models. Here, we report an in vitro 3D tumour model using a hydroxyethyl chitosan/glycidyl methacrylate (HECS-GMA) hydrogel produced by a series of chitosan modifications. Parameters relating to cell morphology, viability, proliferation, and migration were analysed using breast cancer MCF-7 cells. In a xenograft model, secretion of angiogenesis-related growth factors and the anti-angiogenic efficacy of Endostar and Bevacizumab in cells grown in HECS-GMA hydrogels were assessed by immunohistochemistry. Hydroxyethyl chitosan/glycidyl methacrylate hydrogels had a highly porous microstructure, mechanical properties, swelling ratio, and morphology consistent with a 3D tumour model. Compared with a 2D monolayer culture, breast cancer MCF-7 cells residing in the HECS-GMA hydrogels grew as tumour-like clusters in a 3D formation. In a xenograft model, MCF-7 cells cultured in the HECS-GMA hydrogels had increased secretion of angiogenesis-related growth factors. Recombinant human endostatin (Endostar), but not Bevacizumab (Avastin), was an effective anti-angiogenic agent in HECS-GMA hydrogels. The HECS-GMA hydrogel provided a 3D tumour model that mimicked the in vivo cancer microenvironment and supported the growth of MCF7 cells better than traditional tissue culture plates. The HECS

Serum concentrations of nitrite and malondialdehyde as markers of oxidative stress in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors

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Maria Juracy Petrola

2012-01-01

Full Text Available BACKGROUND: Chronic myeloid leukemia is a neoplasm characterized by clonal expansion of hematopoietic progenitor cells resulting from the (9:22(q34,11 translocation. The tyrosine kinase abl fusion protein,the initial leukemogenic event in chronic myeloid leukemia, is constitutively activated thus inducing the production of reactive oxygen species. Of particular relevance is the fact that an increase in reactive oxygen species can facilitate genomic instability and may contribute to disease progression. OBJETIVE: To evaluate oxidative stress by determining the levels of malondialdehyde and nitrite in chronic myeloid leukemia patients under treatment with 1st and 2nd generation tyrosine kinase inhibitors monitored at a referral hospital in Fortaleza, Ceará. METHODS: A cross-sectional study was performed of 64 male and female adults. Patients were stratified according to treatment. The levels of malondialdehyde and nitrite were determined by spectrophotometry. Statistical differences between groups were observed using the Student t-test and Fisher's exact test. The results are expressed as mean ± standard error of mean. The significance level was set for a p-value < 0.05 in all analyses. RESULTS: The results show significantly higher mean concentrations of nitrite and malondialdehyde in chronic myeloid leukemia patients using second-generation tyrosine kinase inhibitors compared to patients on imatinib. Conclusion: It follows that chronic myeloid leukemia patients present higher oxidative activity and that the increases in oxidative damage markers can indicate resistance to 1st generation tyrosine kinase inhibitors.

Tyrosine kinase signalling in breast cancer: Modulation of tyrosine kinase signalling in human breast cancer through altered expression of signalling intermediates

International Nuclear Information System (INIS)

Kairouz, Rania; Daly, Roger J

2000-01-01

The past decade has seen the definition of key signalling pathways downstream of receptor tyrosine kinases (RTKs) in terms of their components and the protein-protein interactions that facilitate signal transduction. Given the strong evidence that links signalling by certain families of RTKs to the progression of breast cancer, it is not surprising that the expression profile of key downstream signalling intermediates in this disease has also come under scrutiny, particularly because some exhibit transforming potential or amplify mitogenic signalling pathways when they are overexpressed. Reflecting the diverse cellular processes regulated by RTKs, it is now clear that altered expression of such signalling proteins in breast cancer may influence not only cellular proliferation (eg Grb2) but also the invasive properties of the cancer cells (eg EMS1/cortactin)

Fragment-based lead discovery of small molecule inhibitors for the EPHA4 receptor tyrosine kinase

NARCIS (Netherlands)

van Linden, O.P.J.; Farenc, C; Zoutman, W.H.; Hameetman, L; Wijtmans, M.; Leurs, R.; Tensen, C.P.; Siegal, G.; de Esch, I.J.P.

2011-01-01

The in silico identification, optimization and crystallographic characterization of a 6,7,8,9-tetrahydro-3H-pyrazolo[3,4-c]isoquinolin-1-amine scaffold as an inhibitor for the EPHA4 receptor tyrosine kinase is described. A database containing commercially available compounds was subjected to an in

Downsizing Treatment with Tyrosine Kinase Inhibitors in Patients with Advanced Gastrointestinal Stromal Tumors Improved Resectability

Science.gov (United States)

Sjölund, Katarina; Andersson, Anna; Nilsson, Erik; Nilsson, Ola; Ahlman, Håkan

2010-01-01

Background Gastrointestinal stromal tumors (GISTs) express the receptor tyrosine kinase KIT. Most GISTs have mutations in the KIT or PDGFRA gene, causing activation of tyrosine kinase. Imatinib, a tyrosine kinase inhibitor (TKI), is the first-line palliative treatment for advanced GISTs. Sunitinib was introduced for patients with mutations not responsive to imatinib. The aim was to compare the survival of patients with high-risk resected GISTs treated with TKI prior to surgery with historical controls and to determine if organ-preserving surgery was facilitated. Methods Ten high-risk GIST-patients had downsizing/adjuvant TKI treatment: nine with imatinib and one with sunitinib. The patients were matched with historical controls (n = 89) treated with surgery alone, from our population-based series (n = 259). Mutational analysis of KIT and PDGFRA was performed in all cases. The progression-free survival was calculated. Results The primary tumors decreased in mean diameter from 20.4 cm to 10.5 cm on downsizing imatinib. Four patients with R0 resection and a period of adjuvant imatinib had no recurrences versus 67% in the historical control group. Four patients with residual liver metastases have stable disease on continuous imatinib treatment after surgery. One patient has undergone reoperation with liver resection. The downsizing treatment led to organ-preserving surgery in nine patients and improved preoperative nutritional status in one patient. Conclusions Downsizing TKI is recommended for patients with bulky tumors with invasion of adjacent organs. Sunitinib can be used for patients in case of imatinib resistance (e.g., wild-type GISTs), underlining the importance of mutational analysis for optimal surgical planning. PMID:20512492

Dual Inhibition of Topoisomerase II and Tyrosine Kinases by the Novel Bis-Fluoroquinolone Chalcone-Like Derivative HMNE3 in Human Pancreatic Cancer Cells.

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Yong-Chao Ma

Full Text Available Both tyrosine kinase and topoisomerase II (TopII are important anticancer targets, and their respective inhibitors are widely used in cancer therapy. However, some combinations of anticancer drugs could exhibit mutually antagonistic actions and drug resistance, which further limit their therapeutic efficacy. Here, we report that HMNE3, a novel bis-fluoroquinolone chalcone-like derivative that targets both tyrosine kinase and TopII, induces tumor cell proliferation and growth inhibition. The viabilities of 6 different cancer cell lines treated with a range of HMNE3 doses were detected using the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay. Cellular apoptosis was determined using Hoechst 33258 fluorescence staining and the terminal deoxynucleotidyl transferase (TdT dUTP nick-end labeling (TUNEL assay. The expression of activated Caspase-3 was examined by immunocytochemistry. The tyrosine kinase activity was measured with a human receptor tyrosine kinase (RTK detection kit using a horseradish peroxidase (HRP-conjugated phosphotyrosine (pY20 antibody as the substrate. The topoisomerase II activity was measured using agarose gel electrophoresis with the DNA plasmid pBR322 as the substrate. The expression levels of the P53, Bax, Bcl-2, Caspase-3, -8, -9, p-cSrc, c-Src and topoisomerase II proteins were detected by western blot analysis. The proliferation of five of the six cancer cell lines was significantly inhibited by HMNE3 at 0.312 to 10 μmol/L in a time- and dose-dependent manner. Treatment of the Capan-1 and Panc-1 cells with 1.6 to 3.2 μM HMNE3 for 48 h significantly increased the percentage of apoptotic cells (P<0.05, and this effect was accompanied by a decrease in tyrosine kinase activity. HMNE3 potentially inhibited tyrosine kinase activity in vitro with an IC50 value of 0.64±0.34 μmol/L in Capan-1 cells and 3.1±0.86 μmol/L in Panc-1 cells. The activity of c-Src was significantly inhibited by HMNE3 in a dose

Binding of a diphosphorylated-ITAM peptide to spleen tyrosine kinase (Syk) induces distal conformational changes : a hydrogen exchange mass spectrometry study

NARCIS (Netherlands)

Catalina, M Isabel; Fischer, Marcel J E; Liskamp, Rob M J; Heck, Albert J R; Dekker, Frank

Structural flexibility plays a crucial role in protein function. To assess whether specific structural changes are associated with the binding of an immunoreceptor tyrosine-based activation motif (ITAM) to the tandem Src homology-2 domains (tSH2) of the spleen tyrosine kinase [EC 2.7.7.112] (Syk),

Anti-Angiogenic Therapeutic Indictors in Breast Cancer

National Research Council Canada - National Science Library

Su, Min-Ying

2003-01-01

This project studies the therapeutic indicators in ant-angiogenic therapy. Every animal with mammary tumor was scheduled to receive a baseline MRI, core biopsy, then followed by 4 treatments with weekly MRI follow...

Vicrostatin - an anti-invasive multi-integrin targeting chimeric disintegrin with tumor anti-angiogenic and pro-apoptotic activities.

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Radu O Minea

2010-06-01

Full Text Available Similar to other integrin-targeting strategies, disintegrins have previously shown good efficacy in animal cancer models with favorable pharmacological attributes and translational potential. Nonetheless, these polypeptides are notoriously difficult to produce recombinantly due to their particular structure requiring the correct pairing of multiple disulfide bonds for biological activity. Here, we show that a sequence-engineered disintegrin (called vicrostatin or VCN can be reliably produced in large scale amounts directly in the oxidative cytoplasm of Origami B E. coli. Through multiple integrin ligation (i.e., alphavbeta3, alphavbeta5, and alpha5beta1, VCN targets both endothelial and cancer cells significantly inhibiting their motility through a reconstituted basement membrane. Interestingly, in a manner distinct from other integrin ligands but reminiscent of some ECM-derived endogenous anti-angiogenic fragments previously described in the literature, VCN profoundly disrupts the actin cytoskeleton of endothelial cells (EC inducing a rapid disassembly of stress fibers and actin reorganization, ultimately interfering with EC's ability to invade and form tubes (tubulogenesis. Moreover, here we show for the first time that the addition of a disintegrin to tubulogenic EC sandwiched in vitro between two Matrigel layers negatively impacts their survival despite the presence of abundant haptotactic cues. A liposomal formulation of VCN (LVCN was further evaluated in vivo in two animal cancer models with different growth characteristics. Our data demonstrate that LVCN is well tolerated while exerting a significant delay in tumor growth and an increase in the survival of treated animals. These results can be partially explained by potent tumor anti-angiogenic and pro-apoptotic effects induced by LVCN.

Synergistic effect of anti-angiogenic herbal composition (Meta-X) in combination with radiotherapy on the inhibition of tumor growth

International Nuclear Information System (INIS)

Han, Young Soo; Song, Jie Young; Yoon, Yeon Sook; Kim, Joon Sik; Park, Byung Young; Lee, Hee Suk; Kim, Min Yung

2004-01-01

Anti-angiogenic composition called Meta-X was made from herbal medicines that are currently used oral drugs for other indications. We examined biochemical properties of Meta-X, and synergistic effect of Meta-X combined with irradiation on the inhibition of tumor growth

Synergistic effect of anti-angiogenic herbal composition (Meta-X) in combination with radiotherapy on the inhibition of tumor growth

Energy Technology Data Exchange (ETDEWEB)

Han, Young Soo; Song, Jie Young; Yoon, Yeon Sook [Korea Institute of Radilolgical and Medical Science, Seoul (Korea, Republic of); Kim, Joon Sik; Park, Byung Young; Lee, Hee Suk; Kim, Min Yung [AngioLab, Seoul (Korea, Republic of)

2004-07-01

Anti-angiogenic composition called Meta-X was made from herbal medicines that are currently used oral drugs for other indications. We examined biochemical properties of Meta-X, and synergistic effect of Meta-X combined with irradiation on the inhibition of tumor growth.

Overall survival after immunotherapy, tyrosine kinase inhibitors and surgery in treatment of metastatic renal cell cancer

DEFF Research Database (Denmark)

de Lichtenberg, Trine Honnens; Hermann, Gregers G.; Rorth, Mikael

2014-01-01

Abstract Objective. The aim of this study was to evaluate overall survival (OS) after treatment of metastatic renal cell carcinoma (mRCC) following the introduction of tyrosine kinase inhibitors (TKIs) and mammalian target of rapamycin (mTOR) inhibitors. Material and methods. One-hundred and forty...

Mutation pattern in the Bruton's tyrosine kinase gene in 26 unrelated patients with X-linked agammaglobulinemia

DEFF Research Database (Denmark)

Vorechovský, I; Luo, L; Hertz, Jens Michael

1997-01-01

Mutation pattern was characterized in the Bruton's tyrosine kinase gene (BTK) in 26 patients with X-linked agammaglobulinemia, the first described immunoglobulin deficiency, and was related to BTK expression. A total of 24 different mutations were identified. Most BTK mutations were found to result...

Functional characterization of autophosphorylation sites of the activated insulin receptor-tyrosine kinase

International Nuclear Information System (INIS)

Flores-Riveros, J.R.; Lane, M.D.

1987-01-01

Insulin receptor, solubilized from 3T3-L1 cellular membranes and then purified, was autophosphorylated with [γ- 32 P]ATP in the absence or presence of insulin. Specific phosphopeptides generated by trypsin digestion of the 32 P-labeled β-subunit were identified and separated by reverse phase HPLC. In the absence of insulin, radioactivity of the phosphopeptides is evenly distributed among four major peaks designated as sites I, II, III and IV, according to their order of elution. This pattern is maintained for at least the first 30 min of autophosphorylation. When the reaction is carried out in the presence of insulin, > 50% of the total 32 P radioactivity is found in site I and the rate of 32 P incorporation into this site is markedly higher than into sites II, III and IV. Maximal activation of tyrosine kinase activity, as estimated by substrate phosphorylation, is coincident with the nearly complete phosphorylation of site I. Delayed activation of previously autophosphorylated receptor by insulin, but not by EGF or IGF-I, produced a similar pattern where phosphorylated site I predominates. These observations indicate that one major insulin-regulated autophosphorylation site in the β-subunit is responsible for activation of the insulin receptor tyrosine kinase. The isolation of this phosphopeptide on a preparative scale and its characterization are now in progress

Tyrosine Kinase Inhibitor Treatment for Newly Diagnosed Chronic Myeloid Leukemia.

Science.gov (United States)

Radich, Jerald P; Mauro, Michael J

2017-08-01

Chronic myeloid leukemia (CML) is a myeloproliferative disorder that accounts for approximately 10% of new cases of leukemia. The introduction of tyrosine kinase inhibitors has led to a reduction in mortalities. Thus, the estimated prevalence of CML is increasing. The National Comprehensive Cancer Network and the European Leukemia Net guidelines incorporate frequent molecular monitoring of the fusion BCR-ABL transcript to ensure that patients reach and keep treatment milestones. Most patients with CML are diagnosed in the chronic phase, and approximately 10% to 30% of these patients will at some time in their course meet definition criteria of resistance to imatinib. Copyright © 2017 Elsevier Inc. All rights reserved.

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

OpenAIRE

Schlaepfer, D D; Hunter, T

1996-01-01

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 req...

Hepatitis C Virus Particle Assembly Involves Phosphorylation of NS5A by the c-Abl Tyrosine Kinase.

Science.gov (United States)

Yamauchi, Shota; Takeuchi, Kenji; Chihara, Kazuyasu; Sun, Xuedong; Honjoh, Chisato; Yoshiki, Hatsumi; Hotta, Hak; Sada, Kiyonao

2015-09-04

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is thought to regulate the replication of viral RNA and the assembly of virus particles in a serine/threonine phosphorylation-dependent manner. However, the host kinases that phosphorylate NS5A have not been fully identified. Here, we show that HCV particle assembly involves the phosphorylation of NS5A by the c-Abl tyrosine kinase. Pharmacological inhibition or knockdown of c-Abl reduces the production of infectious HCV (J6/JFH1) particles in Huh-7.5 cells without markedly affecting viral RNA translation and replication. NS5A is tyrosine-phosphorylated in HCV-infected cells, and this phosphorylation is also reduced by the knockdown of c-Abl. Mutational analysis reveals that NS5A tyrosine phosphorylation is dependent, at least in part, on Tyr(330) (Tyr(2306) in polyprotein numbering). Mutation of this residue to phenylalanine reduces the production of infectious HCV particles but does not affect the replication of the JFH1 subgenomic replicon. These findings suggest that c-Abl promotes HCV particle assembly by phosphorylating NS5A at Tyr(330). © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Ibrutinib Inhibits ERBB Receptor Tyrosine Kinases and HER2-Amplified Breast Cancer Cell Growth.

Science.gov (United States)

Chen, Jun; Kinoshita, Taisei; Sukbuntherng, Juthamas; Chang, Betty Y; Elias, Laurence

2016-12-01

Ibrutinib is a potent, small-molecule Bruton tyrosine kinase (BTK) inhibitor developed for the treatment of B-cell malignancies. Ibrutinib covalently binds to Cys481 in the ATP-binding domain of BTK. This cysteine residue is conserved among 9 other tyrosine kinases, including HER2 and EGFR, which can be targeted. Screening large panels of cell lines demonstrated that ibrutinib was growth inhibitory against some solid tumor cells, including those inhibited by other HER2/EGFR inhibitors. Among sensitive cell lines, breast cancer lines with HER2 overexpression were most potently inhibited by ibrutinib (ibrutinib coincided with downregulation of phosphorylation on HER2 and EGFR and their downstream targets, AKT and ERK. Irreversible inhibition of HER2 and EGFR in breast cancer cells was established after 30-minute incubation above 100 nmol/L or following 2-hour incubation at lower concentrations. Furthermore, ibrutinib inhibited recombinant HER2 and EGFR activity that was resistant to dialysis and rapid dilution, suggesting an irreversible interaction. The dual activity toward TEC family (BTK and ITK) and ERBB family kinases was unique to ibrutinib, as ERBB inhibitors do not inhibit or covalently bind BTK or ITK. Xenograft studies with HER2 + MDA-MB-453 and BT-474 cells in mice in conjunction with determination of pharmacokinetics demonstrated significant exposure-dependent inhibition of growth and key signaling molecules at levels that are clinically achievable. Ibrutinib's unique dual spectrum of activity against both TEC family and ERBB kinases suggests broader applications of ibrutinib in oncology. Mol Cancer Ther; 15(12); 2835-44. ©2016 AACR. ©2016 American Association for Cancer Research.

SAM domain-dependent activity of PfTKL3, an essential tyrosine kinase-like kinase of the human malaria parasite Plasmodium falciparum.

Science.gov (United States)

Abdi, Abdirahman; Eschenlauer, Sylvain; Reininger, Luc; Doerig, Christian

2010-10-01

Over the last decade, several protein kinases inhibitors have reached the market for cancer chemotherapy. The kinomes of pathogens represent potentially attractive targets in infectious diseases. The functions of the majority of protein kinases of Plasmodium falciparum, the parasitic protist responsible for the most virulent form of human malaria, remain unknown. Here we present a thorough characterisation of PfTKL3 (PF13_0258), an enzyme that belongs to the tyrosine kinase-like kinase (TKL) group. We demonstrate by reverse genetics that PfTKL3 is essential for asexual parasite proliferation in human erythrocytes. PfTKL3 is expressed in both asexual and gametocytes stages, and in the latter the protein co-localises with cytoskeleton microtubules. Recombinant PfTKL3 displays in vitro autophosphorylation activity and is able to phosphorylate exogenous substrates, and both activities are dramatically dependent on the presence of an N-terminal "sterile alpha-motif" domain. This study identifies PfTKL3 as a validated drug target amenable to high-throughput screening.

Genome-Wide Identification and Characterization of Tyrosine Kinases in the Silkworm, Bombyx mori

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Songzhen He

2018-03-01

Full Text Available The tyrosine kinases (TKs are important parts of metazoan signaling pathways and play significant roles in cell growth, development, apoptosis and disease. Genome-wide characterization of TKs has been conducted in many metazoans, however, systematic information about this family in Lepidoptera is still lacking. We retrieved 33 TK-encoding genes in silkworm and classified them into 25 subfamilies by sequence analysis, without members in AXL, FRK, PDGFR, STYK1 and TIE subfamilies. Although domain sequences in each subfamily are conserved, TKs in vertebrates tend to be remarkably conserved and stable. Our results of phylogenetic analysis supported the previous conclusion for the second major expansion of TK family. Gene-Ontology (GO analysis revealed that a higher proportion of BmTKs played roles in binding, catalysis, signal transduction, metabolism, biological regulation and response to stimulus, compared to all silkworm genes annotated in GO. Moreover, the expression profile analysis of BmTKs among multiple tissues and developmental stages demonstrated that many genes exhibited stage-specific and/or sex-related expression during embryogenesis, molting and metamorphosis, and that 8 BmTKs presented tissue-specific high expression. Our study provides systematic description of silkworm tyrosine kinases, and may also provide further insights into metazoan TKs and assist future studies addressing their functions.

Antibacterial and EGFR-Tyrosine Kinase Inhibitory Activities of Polyhydroxylated Xanthones from Garcinia succifolia

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Susawat Duangsrisai

2014-11-01

Full Text Available Chemical investigation of the methanol extract of the wood of Garcinia succifolia Kurz (Clusiaceae led to the isolation of 1,5-dihydroxyxanthone (1, 1,7-dihydroxyxanthone (2, 1,3,7-trihydroxyxanthone (3, 1,5,6-trihydroxyxanthone (4, 1,6,7-trihydroxyxanthone (5, and 1,3,6,7-tetrahydroxyxanthone (6. All of the isolated xanthones were evaluated for their antibacterial activity against bacterial reference strains, two Gram-positive (Staphylococcus aureus ATTC 25923, Bacillus subtillis ATCC 6633 and two Gram-negative (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853, and environmental drug-resistant isolates (S. aureus B1, Enteroccoccus faecalis W1, and E. coli G1, as well as for their epidermal growth factor receptor (EGFR of tyrosine kinase inhibitory activity. Only 1,5,6-trihydroxy-(4, 1,6,7-trihydroxy-(5, and 1,3,6,7-tetrahydroxyxanthones (6 exhibited antibacterial activity against Gram-positive bacteria, however none was active against vancomycin-resistant E. faecalis. Additionally, 1,7-dihydroxyxanthone (2 showed synergism with oxacillin, but not with ampicillin. On the other hand, only 1,5-dihydroxyxanthone (1 and 1,7-dihydroxyxanthone (2 were found to exhibit the EGFR-tyrosine kinase inhibitory activity, with IC50 values of 90.34 and 223 nM, respectively.

Melanoma-associated antigen expression and the efficacy of tyrosine kinase inhibitors in head and neck cancer

DEFF Research Database (Denmark)

Hartmann, Stefan; Brands, Roman C; Küchler, Nora

2015-01-01

receptor (EGFR). The efficacy of tyrosine kinase inhibitors (TKI) in the context of melanoma-associated antigens is discussed in the present study. Five human squamous cell carcinoma cell lines were treated with the EGFR TKIs, erlotinib and gefitinib. The efficacy of these agents was measured using...

Soluble TAM receptor tyrosine kinases in rheumatoid arthritis: correlation with disease activity and bone destruction.

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Xu, L; Hu, F; Zhu, H; Liu, X; Shi, L; Li, Y; Zhong, H; Su, Y

2018-04-01

The TAM receptor tyrosine kinases (TAM RTK) are a subfamily of receptor tyrosine kinases, the role of which in autoimmune diseases such as systemic lupus erythematosus has been well explored, while their functions in rheumatoid arthritis (RA) remain largely unknown. In this study, we investigated the role of soluble TAM receptor tyrosine kinases (sAxl/sMer/sTyro3) in patients with RA. A total of 306 RA patients, 100 osteoarthritis (OA) patients and 120 healthy controls (HCs) were enrolled into this study. The serum concentrations of sAxl/sMer/sTyro3 were measured by enzyme-linked immunosorbent assay (ELISA), then the associations between sAxl/sMer/sTyro3 levels and clinical features of RA patients were analysed. We also investigated whether sTyro3 could promote osteoclast differentiation in vitro in RA patients. The results showed that compared with healthy controls (HCs), sTyro3 levels in the serum of RA patients were elevated remarkably and sMer levels were decreased significantly, whereas there was no difference between HCs and RA patients on sAxl levels. The sTyro3 levels were correlated weakly but positively with white blood cells (WBC), immunoglobulin (Ig)M, rheumatoid factor (RF), swollen joint counts, tender joint counts, total sharp scores and joint erosion scores. Conversely, there were no significant correlations between sMer levels and the above indices. Moreover, RA patients with high disease activity also showed higher sTyro3 levels. In-vitro osteoclast differentiation assay showed further that tartrate-resistant acid phosphatase (TRAP) + osteoclasts were increased significantly in the presence of sTyro3. Collectively, our study indicated that serum sTyro3 levels were elevated in RA patients and correlated positively with disease activity and bone destruction, which may serve as an important participant in RA pathogenesis. © 2017 British Society for Immunology.

PROLACTIN-INDUCED TYROSINE PHOSPHORYLATION, ACTIVATION AND RECEPTOR ASSOCIATION OF FOCAL ADHESION KINASE (FAK) IN MAMMARY EPITHELIAL CELLS

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Prolactin-Induced Tyrosine Phosphorylation, Activation and ReceptorAssociation of Focal Adhesion Kinase (FAK) in Mammary Epithelial Cells. Suzanne E. Fenton1 and Lewis G. Sheffield2. 1U.S. Environmental ProtectionAgency, MD-72, Research Triangle Park, NC 27711, and

Kinase profiling of liposarcomas using RNAi and drug screening assays identified druggable targets

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Deepika Kanojia

2017-11-01

Full Text Available Abstract Background Liposarcoma, the most common soft tissue tumor, is understudied cancer, and limited progress has been made in the treatment of metastatic disease. The Achilles heel of cancer often is their kinases that are excellent therapeutic targets. However, very limited knowledge exists of therapeutic critical kinase targets in liposarcoma that could be potentially used in disease management. Methods Large RNAi and small-molecule tyrosine kinase inhibitor screens were performed against the proliferative capacity of liposarcoma cell lines of different subtypes. Each small molecule inhibitor was either FDA approved or in a clinical trial. Results Screening assays identified several previously unrecognized targets including PTK2 and KIT in liposarcoma. We also observed that ponatinib, multi-targeted tyrosine kinase inhibitor, was the most effective drug with anti-growth effects against all cell lines. In vitro assays showed that ponatinib inhibited the clonogenic proliferation of liposarcoma, and this anti-growth effect was associated with apoptosis and cell cycle arrest at the G0/G1 phase as well as a decrease in the KIT signaling pathway. In addition, ponatinib inhibited in vivo growth of liposarcoma in a xenograft model. Conclusions Two large-scale kinase screenings identified novel liposarcoma targets and a FDA-approved inhibitor, ponatinib with clear anti-liposarcoma activity highlighting its potential therapy for treatment of this deadly tumor.

Anti-tumor activity of high-dose EGFR tyrosine kinase inhibitor and sequential docetaxel in wild type EGFR non-small cell lung cancer cell nude mouse xenografts

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Tang, Ning; Zhang, Qianqian; Fang, Shu; Han, Xiao; Wang, Zhehai

2016-01-01

Treatment of non-small-cell lung cancer (NSCLC) with wild-type epidermal growth factor receptor (EGFR) is still a challenge. This study explored antitumor activity of high-dose icotinib (an EGFR tyrosine kinase inhibitor) plus sequential docetaxel against wild-type EGFR NSCLC cells-generated nude mouse xenografts. Nude mice were subcutaneously injected with wild-type EGFR NSCLC A549 cells and divided into different groups for 3-week treatment. Tumor xenograft volumes were monitored and record...

Novel function of transcription factor Nrf2 as an inhibitor of RON tyrosine kinase receptor-mediated cancer cell invasion.

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Thangasamy, Amalraj; Rogge, Jessica; Krishnegowda, Naveen K; Freeman, James W; Ammanamanchi, Sudhakar

2011-09-16

Recepteur d' origine nantais (RON), a tyrosine kinase receptor, is aberrantly expressed in human tumors and promotes cancer cell invasion. RON receptor activation is also associated with resistance to tamoxifen treatment in breast cancer cells. Nrf2 is a positive regulator of cytoprotective genes. Using chromatin immunoprecipitation (ChIP) and site-directed mutagenesis studies of the RON promoter, we identified Nrf2 as a negative regulator of RON gene expression. High Nrf2 and low RON expression was observed in normal mammary tissue whereas high RON and low or undetectable expression of Nrf2 was observed in breast tumors. The Nrf2 inducer sulforaphane (SFN) as well as ectopic Nrf2 expression or knock-down of the Nrf2 negative regulator keap1, which stabilizes Nrf2, inhibited RON expression and invasion of carcinoma cells. Consequently, our studies identified a novel functional role for Nrf2 as a "repressor" and RON kinase as a molecular target of SFN, which mediates the anti-tumor effects of SFN. These results are not limited to breast cancer cells since the Nrf2 inducer SFN stabilized Nrf2 and inhibited RON expression in carcinoma cells from various tumor types.

The tyrosine kinase inhibitor imatinib mesylate suppresses uric acid crystal-induced acute gouty arthritis in mice.

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Laurent L Reber

Full Text Available Gouty arthritis is caused by the deposition of monosodium urate (MSU crystals in joints. Despite many treatment options for gout, there is a substantial need for alternative treatments for patients unresponsive to current therapies. Tyrosine kinase inhibitors have demonstrated therapeutic benefit in experimental models of antibody-dependent arthritis and in rheumatoid arthritis in humans, but to date, the potential effects of such inhibitors on gouty arthritis has not been evaluated. Here we demonstrate that treatment with the tyrosine kinase inhibitor imatinib mesylate (imatinib can suppress inflammation induced by injection of MSU crystals into subcutaneous air pouches or into the ankle joint of wild type mice. Moreover, imatinib treatment also largely abolished the lower levels of inflammation which developed in IL-1R1-/- or KitW-sh/W-sh mice, indicating that this drug can inhibit IL-1-independent pathways, as well as mast cell-independent pathways, contributing to pathology in this model. Imatinib treatment not only prevented ankle swelling and synovial inflammation when administered before MSU crystals but also diminished these features when administrated after the injection of MSU crystals, a therapeutic protocol more closely mimicking the clinical situation in which treatment occurs after the development of an acute gout flare. Finally, we also assessed the efficiency of local intra-articular injections of imatinib-loaded poly(lactic-co-glycolic acid (PLGA nanoparticles in this model of acute gout. Treatment with low doses of this long-acting imatinib:PLGA formulation was able to reduce ankle swelling in a therapeutic protocol. Altogether, these results raise the possibility that tyrosine kinase inhibitors might have utility in the treatment of acute gout in humans.

Insulin receptor binding and tyrosine kinase activity in skeletal muscle from normal pregnant women and women with gestational diabetes

DEFF Research Database (Denmark)

Damm, P.; Handberg, A.; Kühl, C.

1993-01-01

OBJECTIVE: To ascertain whether the decreased glucose tolerance and insulin resistance found in normal and gestational diabetic pregnancy might be associated with changes in insulin receptor function. METHODS: Eight nonpregnant healthy women (nonpregnant controls), eight healthy pregnant women...... (pregnant controls), and eight women with gestational diabetes were investigated. All were non-obese. Muscle biopsies were obtained from the vastus lateralis muscle, and insulin binding and tyrosine kinase activities in partially purified skeletal muscle insulin receptors were studied. The pregnant controls...... with gestational diabetes compared to nonpregnant controls (P pregnant women did not differ from the other two groups. Postpartum, no differences in insulin binding were found between the groups. Basal and maximal tyrosine kinase activities toward the exogenous substrate poly(Glu4Tyr1) were...

Lemur Tyrosine Kinase-3 Suppresses Growth of Prostate Cancer Via the AKT and MAPK Signaling Pathways

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Pengcheng Sun

2017-08-01

Full Text Available Background/Aims: Lemur tyrosine kinase (LMTK-3 is a member of the receptor tyrosine kinase (RTK family. Abnormal expression of LMTK-3 exists in various types of cancers, especially in endocrine-resistant breast cancers; however, the precise level of expression and the biological function in prostate cancer are poorly understood. Methods: In the present study, we determined the expression of LMTK-3 in prostate cancer using immunohistochemistry and Western blotting. We infected PC3 and LNCaP cells with lentivirus-LMTK-3 and observed the biologic characteristics of the PC3 and LNCaP cells in vitro with TUNEL, and migration and invasion assays, respectively. We also established a transplant tumor model of human prostate cancer with infected cells in 15 BALB/c-nu/nu nude mice. Results: LMTK-3 was expressed in prostate epithelial cells. There was a significant decline in the level of LMTK-3 expression in prostate cancers compared to normal tissues. LMTK-3 inhibited PC3 and LNCaP cell growth, migration, and invasion, and induced cell apoptosis in vitro. We also observed that LMTK-3 induced PC3 cell apoptosis in vivo. Further study showed that LMTK-3 inhibited phosphorylation of AKT and ERK, and promoted phosphorylation and activation of p38 kinase and Jun kinase (JNK. Conclusion: Recombinant lentivirus with enhanced expression of LMTK-3 inhibited prostate cancer cell growth and induced apoptosis in vitro and in vivo. AKT and MAPK signaling pathways may contribute to the process.

Alignment independent 3D-QSAR, quantum calculations and molecular docking of Mer specific tyrosine kinase inhibitors as anticancer drugs

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Fereshteh Shiri

2016-03-01

Full Text Available Mer receptor tyrosine kinase is a promising novel cancer therapeutic target in many human cancers, because abnormal activation of Mer has been implicated in survival signaling and chemoresistance. 3D-QSAR analyses based on alignment independent descriptors were performed on a series of 81 Mer specific tyrosine kinase inhibitors. The fractional factorial design (FFD and the enhanced replacement method (ERM were applied and tested as variable selection algorithms for the selection of optimal subsets of molecular descriptors from a much greater pool of such regression variables. The data set was split into 65 molecules as the training set and 16 compounds as the test set. All descriptors were generated by using the GRid INdependent descriptors (GRIND approach. After variable selection, GRIND were correlated with activity values (pIC50 by PLS regression. Of the two applied variable selection methods, ERM had a noticeable improvement on the statistical parameters of PLS model, and yielded a q2 value of 0.77, an rpred2 of 0.94, and a low RMSEP value of 0.25. The GRIND information contents influencing the affinity on Mer specific tyrosine kinase were also confirmed by docking studies. In a quantum calculation study, the energy difference between HOMO and LUMO (gap implied the high interaction of the most active molecule in the active site of the protein. In addition, the molecular electrostatic potential energy at DFT level confirmed results obtained from the molecular docking. The identified key features obtained from the molecular modeling, enabled us to design novel kinase inhibitors.

Alignment independent 3D-QSAR, quantum calculations and molecular docking of Mer specific tyrosine kinase inhibitors as anticancer drugs.

Science.gov (United States)

Shiri, Fereshteh; Pirhadi, Somayeh; Ghasemi, Jahan B

2016-03-01

Mer receptor tyrosine kinase is a promising novel cancer therapeutic target in many human cancers, because abnormal activation of Mer has been implicated in survival signaling and chemoresistance. 3D-QSAR analyses based on alignment independent descriptors were performed on a series of 81 Mer specific tyrosine kinase inhibitors. The fractional factorial design (FFD) and the enhanced replacement method (ERM) were applied and tested as variable selection algorithms for the selection of optimal subsets of molecular descriptors from a much greater pool of such regression variables. The data set was split into 65 molecules as the training set and 16 compounds as the test set. All descriptors were generated by using the GRid INdependent descriptors (GRIND) approach. After variable selection, GRIND were correlated with activity values (pIC50) by PLS regression. Of the two applied variable selection methods, ERM had a noticeable improvement on the statistical parameters of PLS model, and yielded a q (2) value of 0.77, an [Formula: see text] of 0.94, and a low RMSEP value of 0.25. The GRIND information contents influencing the affinity on Mer specific tyrosine kinase were also confirmed by docking studies. In a quantum calculation study, the energy difference between HOMO and LUMO (gap) implied the high interaction of the most active molecule in the active site of the protein. In addition, the molecular electrostatic potential energy at DFT level confirmed results obtained from the molecular docking. The identified key features obtained from the molecular modeling, enabled us to design novel kinase inhibitors.

Bacterial single-stranded DNA-binding proteins are phosphorylated on tyrosine

DEFF Research Database (Denmark)

Mijakovic, Ivan; Petranovic, Dina; Macek, B

2006-01-01

for phosphotyrosine-containing proteins in Streptomyces griseus by immunoaffinity chromatography identified bacterial SSBs as a novel target of bacterial tyrosine kinases. Since genes encoding protein-tyrosine kinases (PTKs) have not been recognized in streptomycetes, and SSBs from Streptomyces coelicolor (Sc......SSB) and Bacillus subtilis (BsSSB) share 38.7% identity, we used a B.subtilis protein-tyrosine kinase YwqD to phosphorylate two cognate SSBs (BsSSB and YwpH) in vitro. We demonstrate that in vivo phosphorylation of B.subtilis SSB occurs on tyrosine residue 82, and this reaction is affected antagonistically...... by kinase YwqD and phosphatase YwqE. Phosphorylation of B.subtilis SSB increased binding almost 200-fold to single-stranded DNA in vitro. Tyrosine phosphorylation of B.subtilis, S.coelicolor and Escherichia coli SSBs occured while they were expressed in E.coli, indicating that tyrosine phosphorylation...

Immuno-Expression of Endoglin and Smooth Muscle Actin in the Vessels of Brain Metastases. Is There a Rational for Anti-Angiogenic Therapy?

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Valeria Barresi

2014-04-01

Full Text Available Despite ongoing clinical trials, the efficacy of anti-angiogenic drugs for the treatment of brain metastases (BM is still questionable. The lower response rate to anti-angiogenic therapy in the presence of BM than in metastatic disease involving other sites suggests that BM may be insensitive to these drugs, although the biological reasons underlining this phenomenon are still to be clarified. With the aim of assessing whether the targets of anti-angiogenic therapies are actually present in BM, in the present study, we analyzed the microvessel density (MVD, a measure of neo-angiogenesis, and the vascular phenotype (mature vs. immature in the tumor tissue of a series of BM derived from different primary tumors. By using immunohistochemistry against endoglin, a specific marker for newly formed vessels, we found that neo-angiogenesis widely varies in BM depending on the site of the primary tumor, as well as on its histotype. According to our results, BM from lung cancer displayed the highest MVD counts, while those from renal carcinoma had the lowest. Then, among BM from lung cancer, those from large cell and adenocarcinoma histotypes had significantly higher MVD counts than those originating from squamous cell carcinoma (p = 0.0043; p = 0.0063. Of note, MVD counts were inversely correlated with the maturation index of the endoglin-stained vessels, reflected by the coverage of smooth muscle actin (SMA positive pericytes (r = −0.693; p < 0.0001. Accordingly, all the endoglin-positive vessels in BM from pulmonary squamous cell carcinoma and renal carcinoma, displayed a mature phenotype, while vessels with an immature phenotype were found in highly vascularized BM from pulmonary large cell and adenocarcinoma. The low MVD and mature phenotype observed in BM from some primary tumors may account for their low sensitivity to anti-angiogenic therapies. Although our findings need to be validated in correlative studies with a clinical response, this should

Increased Tumor Oxygenation and Drug Uptake During Anti-Angiogenic Weekly Low Dose Cyclophosphamide Enhances the Anti-Tumor Effect of Weekly Tirapazamine

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Doloff, J.C.; Khan, N.; Ma, J.; Demidenko, E.; Swartz, H.M.; Jounaidi, Y.

2010-01-01

Metronomic cyclophosphamide treatment is associated with anti-angiogenic activity and is anticipated to generate exploitable hypoxia using hypoxia-activated prodrugs. Weekly administration of tirapazamine (TPZ; 5 mg/kg body weight i.p.) failed to inhibit the growth of 9L gliosarcoma tumors grown s.c. in scid mice. However, the anti-tumor effect of weekly cyclophosphamide (CPA) treatment (140 mg/kg BW i.p.) was substantially enhanced by weekly TPZ administration. An extended tumor free period and increased frequency of tumor eradication without overt toxicity were observed when TPZ was given 3, 4 or 5 days after each weekly CPA treatment. Following the 2nd CPA injection, Electron Paramagnetic Resonance (EPR) Oximetry indicated significant increases in tumor pO2, starting at 48 hr, which further increased after the 3rd CPA injection. pO2 levels were, however, stable in growing untreated tumors. A strong negative correlation (−0.81) between tumor pO2 and tumor volume during 21 days of weekly CPA chemotherapy was observed, indicating increasing tumor pO2 with decreasing tumor volume. Furthermore, CPA treatment resulted in increased tumor uptake of activated CPA. CPA induced increases in VEGF RNA, which reached a maximum on day 1, and in PLGF RNA which was sustained throughout the treatment, while anti-angiogenic host thrombospondin-1 increased dramatically through day 7 post-CPA treatment. Weekly cyclophosphamide treatment was anticipated to generate exploitable hypoxia. However, our findings suggest that weekly CPA treatment induces a functional improvement of tumor vasculature, which is characterized by increased tumor oxygenation and drug uptake in tumors, thus counter-intuitively, benefiting intratumoral activation of TPZ and perhaps other bioreductive drugs. PMID:19754361

TNK2 Tyrosine Kinase as a Novel Therapeutic Target in Triple-Negative Breast Cancer

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2017-10-01

Award Number: W81XWH-15-1-0311 TITLE: TNK2 Tyrosine Kinase as a Novel Therapeutic Target in Triple- Negative Breast Cancer PRINCIPAL...Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Triple-negative breast cancers (TNBCs) represent only 10%-15% of all breast cancers ; however... cancers (TNBC) represent 10-15% of all breast cancers . While significant advances have been made for targeted therapy of ER and HER2-positive breast

CXCL16 is a novel angiogenic factor for human umbilical vein endothelial cells

International Nuclear Information System (INIS)

Zhuge, Xin; Murayama, Toshinori; Arai, Hidenori; Yamauchi, Ryoko; Tanaka, Makoto; Shimaoka, Takeshi; Yonehara, Shin; Kume, Noriaki; Yokode, Masayuki; Kita, Toru

2005-01-01

CXCL16 is a unique chemokine with characteristics as a receptor for phosphatidylserine and oxidized low density lipoproteins in macrophages, and is involved in the accumulation of cellular cholesterol during atherosclerotic lesion development. In this study, we report a new function of CXCL16 as a novel angiogenic factor in human umbilical vein endothelial cells (HUVEC). CXCL16 stimulated proliferation and chemotaxis of HUVEC in a dose-dependent manner, reaching a maximum at 1 nM. CXCL16 also significantly induced tube formation of HUVEC on Matrigel. Further, exposure of HUVEC to CXCL16 led to a time- and dose-dependent activation of mitogen-activated protein kinase (ERK1/2), which was completely inhibited by a mitogen-activated protein kinase kinase inhibitor, PD98059. Proliferation and tube formation in response to CXCL16 were also blocked by the pretreatment with PD98059, but not CXCL16-induced chemotaxis. Thus, our data indicate that CXCL16 may act as a novel angiogenic factor for HUVEC and that ERK is involved as an important signaling molecule to mediate its angiogenic effects

Comparative evaluation of bone marrow cells morpho-functional activity in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors of the first and second generation

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I. O. Zhaleyko

2014-07-01

Full Text Available The efficiency of using the culture techniques of research for monitoring the patient’s response to the treatment by tyrosine kinase inhibitors of the first and second generation is shown. Thus, the functional activity of bone marrow cells in patients having the optimal treatment response to inhibitors of tyrosine kinases was significantly lower compared with patients with the acquired resistance to the drug, and patients who had CML diagnosed for first time. Furthermore, for patients with the optimal response to the nilotinib therapy, numbers of colonies in semi-solid agar in vitro was lower, than in patients with the optimal response to imatinib. When the leukaemic cell clone becomes resistant to tyrosine kinase inhibitors, the prevalence of early cells of granulocyte-macrophage hematopoietic stem cells is observed in CFU culture which can be an important prognostic factor for choosing the appropriate treatment strategy.

Complement receptor-3 negatively regulates the phagocytosis of degenerated myelin through tyrosine kinase Syk and cofilin

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Hadas Smadar

2012-07-01

Full Text Available Abstract Background Intact myelin, which normally surrounds axons, breaks down in Wallerian degeneration following axonal injury and during neurodegenerative diseases such as multiple sclerosis. Clearance of degenerated myelin by phagocytosis is essential since myelin impedes repair and exacerbates damage. CR3 (complement receptor-3 is a principal phagocytic receptor in myelin phagocytosis. We studied how tyrosine kinase Syk (spleen tyrosine kinase and cofilin control phagocytosis of degenerated myelin by CR3 in microglia and macrophages. Syk is a non-receptor tyrosine kinase that CR3 recruits to convey cellular functions. Cofilin is an actin-depolymerizing protein that controls F-actin (filamentous actin remodeling (i.e., disassembly and reassembly by shifting between active unphosphorylated and inactive phosphorylated states. Results Syk was continuously activated during prolonged phagocytosis. Phagocytosis increased when Syk activity and expression were reduced, suggesting that normally Syk down regulates CR3-mediated myelin phagocytosis. Levels of inactive p-cofilin (phosphorylated cofilin decreased transiently during prolonged phagocytosis. In contrast, p-cofilin levels decreased continuously when Syk activity and expression were continuously reduced, suggesting that normally Syk advances the inactive state of cofilin. Observations also revealed inverse relationships between levels of phagocytosis and levels of inactive p-cofilin, suggesting that active unphosphorylated cofilin advances phagocytosis. Active cofilin could advance phagocytosis by promoting F-actin remodeling, which supports the production of membrane protrusions (e.g., filopodia, which, as we also revealed, are instrumental in myelin phagocytosis. Conclusions CR3 both activates and downregulates myelin phagocytosis at the same time. Activation was previously documented. We presently demonstrate that downregulation is mediated through Syk, which advances the inactive

Abelson tyrosine-protein kinase 2 regulates myoblast proliferation and controls muscle fiber length

OpenAIRE

Lee, Jennifer K; Hallock, Peter T; Burden, Steven J

2017-01-01

Muscle fiber length is nearly uniform within a muscle but widely different among different muscles. We show that Abelson tyrosine-protein kinase 2 (Abl2) has a key role in regulating myofiber length, as a loss of Abl2 leads to excessively long myofibers in the diaphragm, intercostal and levator auris muscles but not limb muscles. Increased myofiber length is caused by enhanced myoblast proliferation, expanding the pool of myoblasts and leading to increased myoblast fusion. Abl2 acts in myobla...

Abelson tyrosine-protein kinase 2 Regulates Myoblast Proliferation and Controls Muscle Fiber Length

OpenAIRE

Burden, Steven; Lee, Jennifer

2017-01-01

Muscle fiber length is nearly uniform within a muscle but widely different among muscles. Here, we show that Abelson tyrosine-protein kinase 2 (Abl2) has a key role in regulating myofiber length, as a loss of Abl2 leads to excessively long myofibers in the diaphragm and other muscles. Increased myofiber length is caused by enhanced myoblast proliferation, expanding the pool of available myoblasts and leading to increased myoblast fusion. Abl2 acts in myoblasts, but expansion of the diaphragm ...

Immunological, anti-angiogenic and clinical effects of intratumoral interleukin 12 electrogene therapy combined with metronomic cyclophosphamide in dogs with spontaneous cancer: A pilot study.

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Cicchelero, Laetitia; Denies, Sofie; Vanderperren, Katrien; Stock, Emmelie; Van Brantegem, Leen; de Rooster, Hilde; Sanders, Niek N

2017-08-01

The immunological, anti-angiogenic and clinical effects of metronomic cyclophosphamide and 3 consecutive intratumoral interleukin (IL)-12 gene therapy (electrogene therapy (EGT)) treatments were evaluated in 6 dogs with spontaneous cancer. In all dogs, a decrease in peripheral leukocytes 2 days after IL-12 EGT coincided with erythema and swelling of the tumor. In the tumor, a transient increase in IL-12 levels was measured, whereas a continuous increase in interferon γ (IFNγ) and thrombospondin 1 (TSP-1) were determined in contrast to a continuous decrease in vascular endothelial growth factor (VEGF). In the serum, a transient increase in IL-12 and IL-10 levels were noted in contrast to a transient decrease in VEGF and TSP-1. The treatment resulted in a significant anti-angiogenic effect. Although all primary tumors continued to progress in time, this progression was slower than before treatment according to the contrast-enhanced ultrasound data. Besides the encouraging immunostimulatory and anti-angiogenic effects observed in all dogs we also noticed in 4 out of 6 dogs clinically relevant improvements in quality of life and weight. These results hold great promise for combinatorial strategies of IL-12 EGT and metronomic chemotherapy with conventional antitumor (immuno)therapies. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

The Fyn tyrosine kinase binds Irs-1 and forms a distinct signaling complex during insulin stimulation.

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Sun, X J; Pons, S; Asano, T; Myers, M G; Glasheen, E; White, M F

1996-05-03

Irs-proteins link the receptors for insulin/IGF-1, growth hormones, and several interleukins and interferons to signaling proteins that contain Src homology-2 (SH2). To identify new Irs-1-binding proteins, we screened a mouse embryo expression library with recombinant [32P]Irs-1, which revealed a specific association between p59fyn and Irs-1. The SH2 domain in p59fyn bound to phosphorylated Tyr895 and Tyr1172, which are located in YXX(L/I) motifs. Mutation of p59fyn at the COOH-terminal tyrosine phosphorylation site (Tyr531) enhanced its binding to Irs-1 during insulin stimulation. Binding experiments with various SH2 protein revealed that Grb-2 was largely excluded from Irs-1 complexes containing p59fyn, whereas Grb-2 and p85 occurred in the same Irs-1 complex. By comparison with the insulin receptor, p59fyn kinase phosphorylated a unique cohort of tyrosine residues in Irs-1. These results outline a role for p59fyn or other related Src-kinases during insulin and cytokine signaling.

JAK kinases are required for the bacterial RNA and poly I:C induced tyrosine phosphorylation of PKR

OpenAIRE

Bleiblo, Farag; Michael, Paul; Brabant, Danielle; Ramana, Chilakamarti V; Tai, TC; Saleh, Mazen; Parrillo, Joseph E; Kumar, Anand; Kumar, Aseem

2012-01-01

Discriminating the molecular patterns associated with RNA is central to innate immunity. The protein kinase PKR is a cytosolic sensor involved in the recognition of viral dsRNA and triggering interferon-induced signaling. Here, we identified bacterial RNA as a novel distinct pattern recognized by PKR. We show that the tyrosine phosphorylation of PKR induced by either bacterial RNA or poly I:C is impaired in mutant cells lacking TYK2, JAK1, or JAK2 kinases. PKR was found to be a direct substra...

TAM receptor tyrosine kinase function and the immunopathology of liver disease.

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Mukherjee, S K; Wilhelm, A; Antoniades, C G

2016-06-01

Tyro3, Axl, MERTK (TAM) receptor tyrosine kinases are implicated in the regulation of the innate immune response through clearance of apoptotic cellular debris and control of cytokine signaling cascades. As a result they are pivotal in regulating the inflammatory response to tissue injury. Within the liver, immune regulatory signaling is employed to prevent the overactivation of innate immunity in response to continual antigenic challenge from the gastrointestinal tract. In this review we appraise current understanding of the role of TAM receptor function in the regulation of both innate and adaptive immunity, with a focus on its impact upon hepatic inflammatory pathology. Copyright © 2016 the American Physiological Society.

Primary cilia and coordination of receptor tyrosine kinase (RTK) and transforming growth factor β (TGF-β) signaling

DEFF Research Database (Denmark)

Christensen, Søren Tvorup; Morthorst, Stine Kjær; Mogensen, Johanne Bay

2017-01-01

are at the root of a pleiotropic group of diseases and syndromic disorders called ciliopathies. In this review, we present an overview of primary cilia-mediated regulation of receptor tyrosine kinase (RTK) and transforming growth factor β (TGF-β) signaling. Further, we discuss how defects in the coordination...

Tyrosine kinase inhibitor BIBF1120 ameliorates inflammation, angiogenesis and fibrosis in CCl4-induced liver fibrogenesis mouse model

NARCIS (Netherlands)

Öztürk Akcora, Büsra; Storm, Gerrit; Prakash, Jai; Bansal, Ruchi

2017-01-01

Hepatic fibrosis, a progressive chronic disease mainly caused by hepatitis viral infections, alcohol abuse or metabolic syndrome leading to liver dysfunction and is the growing cause of mortality worldwide. Tyrosine kinase inhibitor BIBF1120 (Nintedanib) has been evaluated in clinical trials for

The effect of riboflavin/UVA cross-linking on anti-degeneration and promoting angiogenic capability of decellularized liver matrix.

Science.gov (United States)

Xiang, Junxi; Liu, Peng; Zheng, Xinglong; Dong, Dinghui; Fan, Shujuan; Dong, Jian; Zhang, Xufeng; Liu, Xuemin; Wang, Bo; Lv, Yi

2017-10-01

Weak mechanical property and unstable degradation rate limited the application of decellularized liver matrix in tissue engineering. The aim of this study was to explore a new method for improving the mechanical properties, anti-degeneration and angiogenic capability of decellularized liver matrix. This was achieved by a novel approach using riboflavin/ultraviolet A treatment to induce collagen cross-linking of decellularized matrix. Histological staining and scanning electron microscope showed that the diameter of cross-linked fibers significantly increased compared with the control group. The average peak load and Young's modulus of decellularized matrix were obviously improved after cross-linking. Then we implanted the modified matrix into the rat hepatic injury model to test the anti-degeneration and angiogenic capability of riboflavin/UVA cross-linked decellularized liver scaffolds in vivo. The results indicated that cross-linked scaffolds degrade more slowly than those in the control group. In the experiment group, average microvessel density in the implanted matrix was higher than that in the control group since the first week after implantation. In conclusion, we initiated the method to improve the biomechanical properties of decellularized liver scaffolds by riboflavin/UVA cross-linking, and more importantly, its improvement on anti-degeneration and angiogenesis was identified. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2662-2669, 2017. © 2017 Wiley Periodicals, Inc.

Promiscuity and selectivity of small-molecule inhibitors across TAM receptor tyrosine kinases in pediatric leukemia.

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Liu, Mao-Hua; Chen, Shi-Bing; Yu, Juan; Liu, Cheng-Jun; Zhang, Xiao-Jing

2017-08-01

The TAM receptor tyrosine kinase family member Mer has been recognized as an attractive therapeutic target for pediatric leukemia. Beside Mer the family contains other two kinases, namely, Tyro3 and Axl, which are highly homologues with Mer and thus most existing small-molecule inhibitors show moderate or high promiscuity across the three kinases. Here, the structural basis and energetic property of selective binding of small-molecule inhibitors to the three kinases were investigated at molecular level. It is found that the selectivity is primarily determined by the size, shape and configuration of kinase's ATP-binding site; the Mer and Axl possess a small, closed active pocket as compared to the bulky, open pocket of Tyro3. The location and conformation of active-site residues of Mer and Axl are highly consistent, suggesting that small-molecule inhibitors generally have a low Mer-over-Axl selectivity and a high Mer-over-Tyro3 selectivity. We demonstrated that the difference in ATP binding potency to the three kinases is also responsible for inhibitor selectivity. We also found that the long-range interactions and allosteric effect arising from rest of the kinase's active site can indirectly influence inhibitor binding and selectivity. Copyright © 2017 Elsevier Inc. All rights reserved.

Suppression of adhesion-induced protein tyrosine phosphorylation decreases invasive and metastatic potentials of B16-BL6 melanoma cells by protein tyrosine kinase inhibitor genistein.

Science.gov (United States)

Yan, C; Han, R

1997-01-01

Protein tyrosine kinase (PTK) appears to be involved in the activation of signaling during cell attachment to and spreading on extracellular matrix (ECM) in the metastatic cascade. To verify the assumption that PTK inhibitors might impair ECM signaling and prevent cancer metastasis, the highly metastatic B16-BL6 mouse melanoma cells were exposed to the PTK inhibitor genistein for 3 days. The ability of the cells to invade through reconstituted basement membrane (Matrigel) and to establish experimental pulmonary metastatic foci in C57BL/6 mice decreased after genistein exposure. The genistein-treated cells were also prevented from attaching to Matrigel and spread extremely poorly on the ECM substratum. Immunoblot analysis showed that tyrosine phosphorylation of a 125-kD protein in response to cell spreading on Matrigel was suppressed in the genistein-treated cells. Adhesion-induced protein tyrosine phosphorylation represents the earlier and specific event in the activation of ECM signaling, so this result implied ECM signaling was impaired in the treated cells. With immunofluorescence microscopy, the adhesion-induced tyrosine phosphorylated proteins were located at the pericytoplasms of well-spread cells, but not at the periphery of poorly spread genistein-treated cells. Therefore, this paper suggests that genistein might impair ECM signaling and subsequently prevent cancer cells from spreading well and invading or establishing metastasis through the suppression of adhesion-induced protein tyrosine phosphorylation. PTKs and adhesion-induced protein tyrosine phosphorylation might play a role in the control of invasion and metastasis.

p56Lck and p59Fyn Regulate CD28 Binding to Phosphatidylinositol 3-Kinase, Growth Factor Receptor-Bound Protein GRB-2, and T Cell-Specific Protein-Tyrosine Kinase ITK: Implications for T-Cell Costimulation

Science.gov (United States)

Raab, Monika; Cai, Yun-Cai; Bunnell, Stephen C.; Heyeck, Stephanie D.; Berg, Leslie J.; Rudd, Christopher E.

1995-09-01

T-cell activation requires cooperative signals generated by the T-cell antigen receptor ξ-chain complex (TCRξ-CD3) and the costimulatory antigen CD28. CD28 interacts with three intracellular proteins-phosphatidylinositol 3-kinase (PI 3-kinase), T cell-specific protein-tyrosine kinase ITK (formerly TSK or EMT), and the complex between growth factor receptor-bound protein 2 and son of sevenless guanine nucleotide exchange protein (GRB-2-SOS). PI 3-kinase and GRB-2 bind to the CD28 phosphotyrosine-based Tyr-Met-Asn-Met motif by means of intrinsic Src-homology 2 (SH2) domains. The requirement for tyrosine phosphorylation of the Tyr-Met-Asn-Met motif for SH2 domain binding implicates an intervening protein-tyrosine kinase in the recruitment of PI 3-kinase and GRB-2 by CD28. Candidate kinases include p56Lck, p59Fyn, ξ-chain-associated 70-kDa protein (ZAP-70), and ITK. In this study, we demonstrate in coexpression studies that p56Lck and p59Fyn phosphorylate CD28 primarily at Tyr-191 of the Tyr-Met-Asn-Met motif, inducing a 3- to 8-fold increase in p85 (subunit of PI 3-kinase) and GRB-2 SH2 binding to CD28. Phosphatase digestion of CD28 eliminated binding. In contrast to Src kinases, ZAP-70 and ITK failed to induce these events. Further, ITK binding to CD28 was dependent on the presence of p56Lck and is thus likely to act downstream of p56Lck/p59Fyn in a signaling cascade. p56Lck is therefore likely to be a central switch in T-cell activation, with the dual function of regulating CD28-mediated costimulation as well as TCR-CD3-CD4 signaling.

Novel PI3K/AKT targeting anti-angiogenic activities of 4-vinylphenol, a new therapeutic potential of a well-known styrene metabolite.

Science.gov (United States)

Yue, Grace Gar-Lee; Lee, Julia Kin-Ming; Kwok, Hin-Fai; Cheng, Ling; Wong, Eric Chun-Wai; Jiang, Lei; Yu, Hua; Leung, Hoi-Wing; Wong, Yuk-Lau; Leung, Ping-Chung; Fung, Kwok-Pui; Lau, Clara Bik-San

2015-06-08

The pneumo- and hepato-toxicity of 4-vinylphenol (4VP), a styrene metabolite, has been previously reported. Nevertheless, the present study reported the novel anti-angiogenic activities of 4VP which was firstly isolated from the aqueous extract of a Chinese medicinal herb Hedyotis diffusa. Our results showed that 4VP at non-toxic dose effectively suppressed migration, tube formation, adhesion to extracellular matrix proteins, as well as protein and mRNA expressions of metalloproteinase-2 of human endothelial cells (HUVEC and HMEC-1). Investigation of the signal transduction revealed that 4VP down-regulated PI3K/AKT and p38 MAPK. Besides, 4VP interfered with the phosphorylation of ERK1/2, the translocation and expression of NFkappaB. In zebrafish embryo model, the new blood vessel growth was significantly blocked by 4VP (6.25-12.5 μg/mL medium). The VEGF-induced blood vessel formation in Matrigel plugs in C57BL/6 mice was suppressed by 4VP (20-100 μg/mL matrigel). In addition, the blood vessel number and tumor size were reduced by intraperitoneal 4VP (0.2-2 mg/kg) in 4T1 breast tumor-bearing BALB/c mice, with doxorubicin as positive control. Together, the in vitro and in vivo anti-angiogenic activities of 4VP were demonstrated for the first time. These findings suggest that 4VP has great potential to be further developed as an anti-angiogenic agent.

Rapid Phospho-Turnover by Receptor Tyrosine Kinases Impacts Downstream Signaling and Drug Binding

OpenAIRE

Kleiman, Laura B.; Maiwald, Thomas; Conzelmann, Holger; Lauffenburger, Douglas A.; Sorger, Peter K.

2011-01-01

Epidermal growth factor receptors (ErbB1–4) are oncogenic receptor tyrosine kinases (RTKs) that regulate diverse cellular processes. In this study, we combine measurement and mathematical modeling to quantify phospho-turnover at ErbB receptors in human cells and to determine the consequences for signaling and drug binding. We find that phosphotyrosine residues on ErbB1 have half-lives of a few seconds and therefore turn over 100–1000 times in the course of a typical immediate-early response t...

X-linked agammaglobulinemia - first case with Bruton tyrosine kinase mutation from Pakistan.

Science.gov (United States)

Zaidi, Samreen Kulsom; Qureshi, Sonia; Qamar, Farah Naz

2017-03-01

X-linked agammaglobulinemia (XLA) is a primary immunodeficiency with more than 600 mutations in Bruton tyrosine kinase (Bkt) gene which are responsible for early-onset agammaglobulinemia and repeated infections. Herein we present a case of a 3-year-old boy with history of repeated diarrhoea and an episode of meningoencephalitis with hemiplegia. The workup showed extremely low levels of immunoglobulin with low CD+19 cells. Genetic analysis showed Btk mutation 18 c.1883delCp.T628fs. To the best of our knowledge this is the first report of a case of XLA confirmed by molecular technique from Pakistan.

Phosphorylation of SLP-76 by the ZAP-70 protein-tyrosine kinase is required for T-cell receptor function.

Science.gov (United States)

Bubeck Wardenburg, J; Fu, C; Jackman, J K; Flotow, H; Wilkinson, S E; Williams, D H; Johnson, R; Kong, G; Chan, A C; Findell, P R

1996-08-16

Two families of tyrosine kinases, the Src and Syk families, are required for T-cell receptor activation. While the Src kinases are responsible for phosphorylation of receptor-encoded signaling motifs and for up-regulation of ZAP-70 activity, the downstream substrates of ZAP-70 are unknown. Evidence is presented herein that the Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76) is a substrate of ZAP-70. Phosphorylation of SLP-76 is diminished in T cells that express a catalytically inactive ZAP-70. Moreover, SLP-76 is preferentially phosphorylated by ZAP-70 in vitro and in heterologous cellular systems. In T cells, overexpression of wild-type SLP-76 results in a hyperactive receptor, while expression of a SLP-76 molecule that is unable to be tyrosine-phosphorylated attenuates receptor function. In addition, the SH2 domain of SLP-76 is required for T-cell receptor function, although its role is independent of the ability of SLP-76 to undergo tyrosine phosphorylation. As SLP-76 interacts with both Grb2 and phospholipase C-gamma1, these data indicate that phosphorylation of SLP-76 by ZAP-70 provides an important functional link between the T-cell receptor and activation of ras and calcium pathways.

Molecular mechanism of 17-allylamino-17-demethoxygeldanamycin (17-AAG)-induced AXL receptor tyrosine kinase degradation.

Science.gov (United States)

Krishnamoorthy, Gnana Prakasam; Guida, Teresa; Alfano, Luigi; Avilla, Elvira; Santoro, Massimo; Carlomagno, Francesca; Melillo, Rosa Marina

2013-06-14

The receptor tyrosine kinase AXL is overexpressed in many cancer types including thyroid carcinomas and has well established roles in tumor formation and progression. Proper folding, maturation, and activity of several oncogenic receptor tyrosine kinases require HSP90 chaperoning. HSP90 inhibition by the antibiotic geldanamycin or its derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) causes destabilization of its client proteins. Here we show that AXL is a novel client protein of HSP90. 17-AAG induced a time- and dose-dependent down-regulation of endogenous or ectopically expressed AXL protein, thereby inhibiting AXL-mediated signaling and biological activity. 17-AAG-induced AXL down-regulation specifically affected fully glycosylated mature receptor present on cell membrane. By using biotin and [(35)S]methionine labeling, we showed that 17-AAG caused depletion of membrane-localized AXL by mediating its degradation in the intracellular compartment, thus restricting its exposure on the cell surface. 17-AAG induced AXL polyubiquitination and subsequent proteasomal degradation; under basal conditions, AXL co-immunoprecipitated with HSP90. Upon 17-AAG treatment, AXL associated with the co-chaperone HSP70 and the ubiquitin E3 ligase carboxyl terminus of HSC70-interacting protein (CHIP). Overexpression of CHIP, but not of the inactive mutant CHIP K30A, induced accumulation of AXL polyubiquitinated species upon 17-AAG treatment. The sensitivity of AXL to 17-AAG required its intracellular domain because an AXL intracellular domain-deleted mutant was insensitive to the compound. Active AXL and kinase-dead AXL were similarly sensitive to 17-AAG, implying that 17-AAG sensitivity does not require receptor phosphorylation. Overall our data elucidate the molecular basis of AXL down-regulation by HSP90 inhibitors and suggest that HSP90 inhibition in anticancer therapy can exert its effect through inhibition of multiple kinases including AXL.

Molecular Mechanism of 17-Allylamino-17-demethoxygeldanamycin (17-AAG)-induced AXL Receptor Tyrosine Kinase Degradation*

Science.gov (United States)

Krishnamoorthy, Gnana Prakasam; Guida, Teresa; Alfano, Luigi; Avilla, Elvira; Santoro, Massimo; Carlomagno, Francesca; Melillo, Rosa Marina

2013-01-01

The receptor tyrosine kinase AXL is overexpressed in many cancer types including thyroid carcinomas and has well established roles in tumor formation and progression. Proper folding, maturation, and activity of several oncogenic receptor tyrosine kinases require HSP90 chaperoning. HSP90 inhibition by the antibiotic geldanamycin or its derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) causes destabilization of its client proteins. Here we show that AXL is a novel client protein of HSP90. 17-AAG induced a time- and dose-dependent down-regulation of endogenous or ectopically expressed AXL protein, thereby inhibiting AXL-mediated signaling and biological activity. 17-AAG-induced AXL down-regulation specifically affected fully glycosylated mature receptor present on cell membrane. By using biotin and [35S]methionine labeling, we showed that 17-AAG caused depletion of membrane-localized AXL by mediating its degradation in the intracellular compartment, thus restricting its exposure on the cell surface. 17-AAG induced AXL polyubiquitination and subsequent proteasomal degradation; under basal conditions, AXL co-immunoprecipitated with HSP90. Upon 17-AAG treatment, AXL associated with the co-chaperone HSP70 and the ubiquitin E3 ligase carboxyl terminus of HSC70-interacting protein (CHIP). Overexpression of CHIP, but not of the inactive mutant CHIP K30A, induced accumulation of AXL polyubiquitinated species upon 17-AAG treatment. The sensitivity of AXL to 17-AAG required its intracellular domain because an AXL intracellular domain-deleted mutant was insensitive to the compound. Active AXL and kinase-dead AXL were similarly sensitive to 17-AAG, implying that 17-AAG sensitivity does not require receptor phosphorylation. Overall our data elucidate the molecular basis of AXL down-regulation by HSP90 inhibitors and suggest that HSP90 inhibition in anticancer therapy can exert its effect through inhibition of multiple kinases including AXL. PMID:23629654

PTP1B Inhibition Causes Rac1 Activation by Enhancing Receptor Tyrosine Kinase Signaling

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Ayako Tsuchiya

2014-04-01

Full Text Available Background/Aims: The present study investigated the signaling pathway underlying Rac1 activation induced by the linoleic acid derivative 8-[2-(2-pentyl-cyclopropylmethyl-cyclopropyl]-octanoic acid (DCP-LA. Methods: Activity of protein tyrosine phosphatase 1B (PTP1B was assayed under cell-free conditions. Western blot was carried out to quantify phosphorylation of insulin receptor substrate-1 (IRS-1 and Akt in PC-12 cells. Rac1 activity was monitored in the föerster resonance energy transfer (FRET analysis using living and fixed PC-12 cells. Results: DCP-LA markedly suppressed PTP1B activity in a concentration (100 pM-100 µM-dependent manner. In the DCP-LA binding assay, fluorescein-conjugated DCP-LA produced a single fluorescent signal band at 60 kDa, corresponding to the molecule of PTP1B, and the signal was attenuated or abolished by co-treatment or pretreatment with non-conjugated DCP-LA. DCP-LA significantly enhanced nerve growth factor (NGF-stimulated phosphorylation of IRS-1 at Tyr1222 and Akt1/2 at Thr308/309 and Ser473/474 in PC-12 cells. In the FRET analysis, DCP-LA significantly enhanced NGF-stimulated Rac1 activation, which is abrogated by the phosphatidylinositol 3 kinase (PI3K inhibitor wortmannin, the 3-phosphoinositide-dependent protein kinase-1 (PDK1 inhibitor BX912, or the Akt inhibitor MK2206. Conclusion: The results of the present study show that DCP-LA-induced PTP1B inhibition, possibly through its direct binding, causes Rac1 activation by enhancing a pathway along a receptor tyrosine kinase (RTK/IRS-1/PI3K/Akt/Rac1 axis.

Role of tyrosine phosphatase inhibitors in cancer treatment with emphasis on SH2 domain-containing tyrosine phosphatases (SHPs)

NARCIS (Netherlands)

Irandoust, Mahban; van den Berg, Timo K.; Kaspers, Gertjan J. L.; Cloos, Jacqueline

2009-01-01

Protein tyrosine phosphorylation is one of the key mechanisms involved in signal transduction pathways. This modification is regulated by concerted action of protein tyrosine phosphatases and protein tyrosine kinases. Deregulation of either of these key regulators lead to abnormal cellular

Src inhibitor herbimycin A prevents 132.7 kDa tyrosine phosphatase activity in Ramos Burkitt's lymphoma B cell line

International Nuclear Information System (INIS)

Hristov, K.; Mitev, V.; Knox, K.

2006-01-01

Reversible tyrosine phosphorylation, regulation of expression and proteolytic cleavage control tyrosine phosphatase contribution for the signalling pathways of B-cell antigen receptor (BCR), and CD40 during B cell selection. We used Ramos-BL B cell line to determine whether BCR and CD40 stimulation, or inhibition of the Src - tyrosine kinase, tyrosine phosphatase and caspase activity have an effect on the tyrosine phosphatase activities determined on in-gel phosphatase assay. The tyrosine phosphatase activities present in whole cell lysates of Ramos-BL B cells following treatment with 20 μg/ml anti-IgM, 1 μg/ml anti-CD40, 10 μM herbimycin A, 178 μM vanadate,100 μM phenylarsine oxide and 10 μM zVAD-fmk were detected with an in-gel phosphatase assay. Seven major tyrosine phosphatase activities with approximate molecular weight of 132.7, 63.9, 60.3, 54.2, 49.7, 44.6, and 39 kDa are present in whole cell lysates of Ramos-BL B cells. Treatment with Src-PTK inhibitor herbimycin A prevents 132.7 kDa tyrosine phosphatase activity. We conclude that the catalytic activity of Src-PTK in Ramos-BL B cells is critical for the presence of this 132.7 kDa tyrosine phosphatase activity. (authors)

Tyrosine and aurora kinase inhibitors diminish transport function of multidrug resistance-associated protein (MRP 4 and breast cancer resistance protein (BCRP

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Rhiannon N. Hardwick

2016-12-01

Full Text Available Tyrosine and aurora kinases are important effectors in signal transduction pathways that are often involved in aberrant cancer cell growth. Tyrosine (TKI and aurora (AKI kinase inhibitors are anti-cancer agents specifically designed to target such signaling pathways through TKI/AKI binding to the ATP-binding pocket of kinases thereby leading to diminished kinase activity. Some TKIs have been identified as inhibitors of ATP-binding cassette (ABC transporters such as P-glycoprotein and breast cancer resistance protein (BCRP, which are commonly upregulated in malignant cells. TKI/AKIs have been investigated as ABC transporter inhibitors in order to facilitate the accumulation of concomitantly administered chemo-therapeutics within cancer cells. However, ABC transporters are prominently expressed in the liver and other eliminating organs, and their inhibition has been linked to intracellular accumulation of drugs, altered disposition, and toxicity. The potential for TKIs/AKIs to inhibit other important hepatic efflux transporters, particularly multidrug resistance-associated proteins (MRPs, remains unknown. The aim of the current study was to compare the inhibitory potency of 20 selected TKI/AKIs against MRP4 and BCRP through the use of inverted membrane vesicle assays. Relative IC50 values were estimated by determining TKI/AKI inhibition of MRP4-mediated [3H]-dehydroepiandrosterone sulfate uptake and BCRP-mediated [3H]-estrone sulfate uptake. To provide insight to the clinical relevance of TKI/AKI inhibition of ABC efflux transporters, the ratio of the steady-state maximum total plasma concentration (Css to the IC50 for each compound was calculated with Css/IC50 ratio >0.1 deemed potentially clinically relevant. Such analysis identified several potentially clinically relevant inhibitors of MRP4: alisertib, danusertib, erlotinib, lapatinib, neratinib, nilotinib, pazopanib, sorafenib, and tozasertib. The potentially clinically relevant inhibition of

Crystal structure of the EphA4 protein tyrosine kinase domain in the apo-and dasantinib-bound state

NARCIS (Netherlands)

Farenc, C; Celie, C; Tensen, P.H.N; de Esch, I.J.P.; Siegal, C.P.

2011-01-01

The Eph family of receptor tyrosine kinases regulates diverse cellular processes while the over-expression of a member of this family, EphA4, has been reported in a variety of malignant carcinomas. To gain insight into molecular mechanisms and to facilitate structure-based inhibitor design, we

Molecularly characterized solvent extracts and saponins from Polygonum hydropiper L show high anti-angiogenic, anti-tumor, brine shrimp and fibroblast NIH/3T3 cell line cytotoxicity

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Muhammad eAyaz

2016-03-01

Full Text Available Polygonum hydropiper is used as anti-cancer and anti-rheumatic agent in folk medicine. This study was designed to investigate the anti-angiogenic, anti-tumor and cytotoxic potentials of different solvent extracts and isolated saponins. Samples were analyzed using GC, GC-MS to identify major and bioactive compounds. Quantitation of antiangiogenesis for the plant's samples including methanolic extract (Ph.Cr, its subsequent fractions; n-hexane (Ph.Hex, chloroform (Ph.Chf, ethyl acetate (Ph.EtAc, n-Butanol (Ph.Bt, aqueous (Ph.Aq, saponins (Ph.Sp were performed using the chick embryo chorioallantoic membrane (CAM assay. Potato disc anti-tumor assay was performed on Agrobacterium tumefaciens containing tumor inducing plasmid. Cytotoxicity was performed on Artemia salina and mouse embryonic fibroblast NIH/3T3 cell line using brine shrimps and MTT cells viability assays. The GC-MS analysis of Ph.Cr, Ph.Hex, Ph.Chf, Ph.Bt and Ph.EtAc identified 126, 124, 153, 131 and 164 compounds respectively. In anti-angiogenic assay, Ph.Chf, Ph.Sp, Ph.EtAc and Ph.Cr exhibited highest activity with IC50 of 28.65, 19.21, 88.75 and 461.53 µg/ml respectively. In anti-tumor assay, Ph.Sp, Ph.Chf, Ph.EtAc and Ph.Cr were most potent with IC50 of 18.39, 73.81, 217.19 and 342.53 µg/ml respectively. In MTT cells viability assay, Ph.Chf, Ph.EtAc, Ph.Sp were most active causing 79.00, 72.50 and 71.50% cytotoxicity respectively at 1000 µg/ml with the LD50 of 140, 160 and 175 µg/ml respectively. In overall study, Ph.Chf and Ph.Sp have shown overwhelming results which signifies their potentials as sources of therapeutic agents against cancer.

Reduced angiogenic factor expression in intrauterine fetal growth restriction using semiquantitative immunohistochemistry and digital image analysis.

Science.gov (United States)

Alahakoon, Thushari I; Zhang, Weiyi; Arbuckle, Susan; Zhang, Kewei; Lee, Vincent

2018-05-01

To localize, quantify and compare angiogenic factors, vascular endothelial growth factor (VEGF), placental growth factor (PlGF), as well as their receptors fms-like tyrosine kinase receptor (Flt-1) and kinase insert domain receptor (KDR) in the placentas of normal pregnancy and complications of preeclampsia (PE), intrauterine fetal growth restriction (IUGR) and PE + IUGR. In a prospective cross-sectional case-control study, 30 pregnant women between 24-40 weeks of gestation, were recruited into four clinical groups. Representative placental samples were stained for VEGF, PlGF, Flt-1 and KDR. Analysis was performed using semiquantitative methods and digital image analysis. The overall VEGF and Flt-1 were strongly expressed and did not show any conclusive difference in the expression between study groups. PlGF and KDR were significantly reduced in expression in the placentas from pregnancies complicated by IUGR compared with normal and preeclamptic pregnancies. The lack of PlGF and KDR may be a cause for the development of IUGR and may explain the loss of vasculature and villous architecture in IUGR. Automated digital image analysis software is a viable alternative method to the manual reading of placental immunohistochemical staining. © 2018 Japan Society of Obstetrics and Gynecology.

Cardiac angiogenic imbalance leads to peripartum cardiomyopathy.

Science.gov (United States)

Patten, Ian S; Rana, Sarosh; Shahul, Sajid; Rowe, Glenn C; Jang, Cholsoon; Liu, Laura; Hacker, Michele R; Rhee, Julie S; Mitchell, John; Mahmood, Feroze; Hess, Philip; Farrell, Caitlin; Koulisis, Nicole; Khankin, Eliyahu V; Burke, Suzanne D; Tudorache, Igor; Bauersachs, Johann; del Monte, Federica; Hilfiker-Kleiner, Denise; Karumanchi, S Ananth; Arany, Zoltan

2012-05-09

Peripartum cardiomyopathy (PPCM) is an often fatal disease that affects pregnant women who are near delivery, and it occurs more frequently in women with pre-eclampsia and/or multiple gestation. The aetiology of PPCM, and why it is associated with pre-eclampsia, remain unknown. Here we show that PPCM is associated with a systemic angiogenic imbalance, accentuated by pre-eclampsia. Mice that lack cardiac PGC-1α, a powerful regulator of angiogenesis, develop profound PPCM. Importantly, the PPCM is entirely rescued by pro-angiogenic therapies. In humans, the placenta in late gestation secretes VEGF inhibitors like soluble FLT1 (sFLT1), and this is accentuated by multiple gestation and pre-eclampsia. This anti-angiogenic environment is accompanied by subclinical cardiac dysfunction, the extent of which correlates with circulating levels of sFLT1. Exogenous sFLT1 alone caused diastolic dysfunction in wild-type mice, and profound systolic dysfunction in mice lacking cardiac PGC-1α. Finally, plasma samples from women with PPCM contained abnormally high levels of sFLT1. These data indicate that PPCM is mainly a vascular disease, caused by excess anti-angiogenic signalling in the peripartum period. The data also explain how late pregnancy poses a threat to cardiac homeostasis, and why pre-eclampsia and multiple gestation are important risk factors for the development of PPCM.

1-Methoxy-agroclavine from Penicillium sp. WC75209, a novel inhibitor of the Lck tyrosine kinase.

Science.gov (United States)

Padmanabha, R; Shu, Y Z; Cook, L S; Veitch, J A; Donovan, M; Lowe, S; Huang, S; Pirnik, D; Manly, S P

1998-03-17

A high-throughput screen was developed and implemented to identify inhibitors of the Lck tyrosine kinase. This report describes the identification of a specific inhibitor of this enzyme from the solid fermentation culture of the Penicillium sp., WC75209. The active compound was isolated and structurally characterized as 1-methoxy-5R, 10S-agroclavine, a new member of the ergot alkaloid family.

The bruton tyrosine kinase inhibitor ibrutinib (PCI-32765) blocks hairy cell leukaemia survival, proliferation and B cell receptor signalling: a new therapeutic approach.

Science.gov (United States)

Sivina, Mariela; Kreitman, Robert J; Arons, Evgeny; Ravandi, Farhad; Burger, Jan A

2014-07-01

B cell receptor (BCR) signalling plays a critical role in the progression of several B-cell malignancies, but its role in hairy cell leukaemia (HCL) is ambiguous. Bruton tyrosine kinase (BTK), a key player in BCR signalling, as well as B cell migration and adhesion, can be targeted with ibrutinib, a selective, irreversible BTK inhibitor. We analysed BTK expression and function in HCL and analysed the effects of ibrutinib on HCL cells. We demonstrated uniform BTK protein expression in HCL cells. Ibrutinib significantly inhibited HCL proliferation and cell cycle progression. Accordingly, ibrutinib also reduced HCL cell survival after BCR triggering with anti-immunoglobulins and abrogated the activation of kinases downstream of the BCR (PI3K and MAPK). Ibrutinib also inhibited BCR-dependent secretion of the chemokines CCL3 and CCL4 by HCL cells. Interestingly, ibrutinib inhibited also CXCL12-induced signalling, a key pathway for bone marrow homing. Collectively, our data support the clinical development of ibrutinib in patients with HCL. © 2014 John Wiley & Sons Ltd.

Identification of SH2-Bbeta as a substrate of the tyrosine kinase JAK2 involved in growth hormone signaling.

OpenAIRE

Rui, L; Mathews, L S; Hotta, K; Gustafson, T A; Carter-Su, C

1997-01-01

Activation of the tyrosine kinase JAK2 is an essential step in cellular signaling by growth hormone (GH) and multiple other hormones and cytokines. Murine JAK2 has a total of 49 tyrosines which, if phosphorylated, could serve as docking sites for Src homology 2 (SH2) or phosphotyrosine binding domain-containing signaling molecules. Using a yeast two-hybrid screen of a rat adipocyte cDNA library, we identified a splicing variant of the SH2 domain-containing protein SH2-B, designated SH2-Bbeta,...

Effects of Src Kinase Inhibition on Expression of Protein Tyrosine Phosphatase 1B after Brain Hypoxia in a Piglet Animal Model

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Dimitrios Angelis

2017-01-01

Full Text Available Background. Protein tyrosine phosphatases (PTPs in conjunction with protein tyrosine kinases (PTKs regulate cellular processes by posttranslational modifications of signal transduction proteins. PTP nonreceptor type 1B (PTP-1B is an enzyme of the PTP family. We have previously shown that hypoxia induces an increase in activation of a class of nonreceptor PTK, the Src kinases. In the present study, we investigated the changes that occur in the expression of PTP-1B in the cytosolic component of the brain of newborn piglets acutely after hypoxia as well as long term for up to 2 weeks. Methods. Newborn piglets were divided into groups: normoxia, hypoxia, hypoxia followed by 1 day and 15 days in FiO2 0.21, and hypoxia pretreated with Src kinase inhibitor PP2, prior to hypoxia followed by 1 day and 15 days. Hypoxia was achieved by providing 7% FiO2 for 1 hour and PTP-1B expression was measured via immunoblotting. Results. PTP-1B increased posthypoxia by about 30% and persisted for 2 weeks while Src kinase inhibition attenuated the expected PTP-1B-increased expression. Conclusions. Our study suggests that Src kinase mediates a hypoxia-induced increased PTP-1B expression.

A biomimetic collagen derived peptide exhibits anti-angiogenic activity in triple negative breast cancer.

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Elena V Rosca

Full Text Available We investigated the application of a mimetic 20 amino acid peptide derived from type IV collagen for treatment of breast cancer. We showed that the peptide induced a decrease of proliferation, adhesion, and migration of endothelial and tumor cells in vitro. We also observed an inhibition of triple negative MDA-MB-231 xenograft growth by 75% relative to control when administered intraperitoneally for 27 days at 10 mg/kg. We monitored in vivo the changes in vascular properties throughout the treatment using MRI and found that the vascular volume and permeability surface area product decreased significantly. The treatment also resulted in an increase of caspase-3 activity and in a reduction of microvascular density. The multiple mode of action of this peptide, i.e., anti-angiogenic, and anti-tumorigenic, makes it a viable candidate as a therapeutic agent as a monotherapy or in combination with other compounds.

Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases

Energy Technology Data Exchange (ETDEWEB)

Chen, Ying-Nan P.; LaMarche, Matthew J.; Chan, Ho Man; Fekkes, Peter; Garcia-Fortanet, Jorge; Acker, Michael G.; Antonakos, Brandon; Chen, Christine Hiu-Tung; Chen, Zhouliang; Cooke, Vesselina G.; Dobson, Jason R.; Deng, Zhan; Fei, Feng; Firestone, Brant; Fodor, Michelle; Fridrich, Cary; Gao, Hui; Grunenfelder, Denise; Hao, Huai-Xiang; Jacob, Jaison; Ho, Samuel; Hsiao, Kathy; Kang, Zhao B.; Karki, Rajesh; Kato, Mitsunori; Larrow, Jay; La Bonte, Laura R.; Lenoir, Francois; Liu, Gang; Liu, Shumei; Majumdar, Dyuti; Meyer, Matthew J.; Palermo, Mark; Perez, Lawrence; Pu, Minying; Price, Edmund; Quinn, Christopher; Shakya, Subarna; Shultz, Michael D.; Slisz, Joanna; Venkatesan, Kavitha; Wang, Ping; Warmuth, Markus; Williams, Sarah; Yang, Guizhi; Yuan, Jing; Zhang, Ji-Hu; Zhu, Ping; Ramsey, Timothy; Keen, Nicholas J.; Sellers, William R.; Stams, Travis; Fortin , Pascal D. (Novartis)

2016-06-29

The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase1. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma1, 2, 3, 4, 5. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS–ERK signalling pathway2, 3. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways6, 7. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy8, 9. Here we report the discovery of a highly potent (IC50 = 0.071 μM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RAS–ERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers.

Regulation of Src family kinases involved in T cell receptor signaling by protein-tyrosine phosphatase CD148

Czech Academy of Sciences Publication Activity Database

Štěpánek, Ondřej; Kalina, T.; Dráber, Peter; Skopcová, Tereza; Svojgr, K.; Angelisová, Pavla; Hořejší, Václav; Weiss, A.; Brdička, Tomáš

2011-01-01

Roč. 286, č. 25 (2011), s. 22101-22112 ISSN 0021-9258 R&D Projects: GA MŠk 2B06064; GA MŠk 1M0506 Institutional research plan: CEZ:AV0Z50520514 Keywords : CD148 * tyrosine phosphatase * Src family kinases Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.773, year: 2011

DETECTION AND PURIFICATION OF TYROSINE-SULFATED PROTEINS USING A NOVEL ANTI-SULFOTYROSINE MONOCLONAL ANTIBODY*

Science.gov (United States)

Hoffhines, Adam J.; Damoc, Eugen; Bridges, Kristie G.; Leary, Julie A.; Moore, Kevin L.

2006-01-01

Protein-tyrosine O-sulfation is a post-translational modification mediated by one of two Golgi tyrosylprotein sulfotransferases (TPST-1 & TPST-2) that catalyze the transfer of sulfate to tyrosine residues in secreted and transmembrane proteins. Tyrosine sulfation plays a role in protein-protein interactions in several well-defined systems. Although dozens of tyrosine-sulfated proteins are known, many more are likely to exist and await description. Advancing our understanding of the importance of tyrosine sulfation in biological systems requires the development of new tools for the detection and study of tyrosine-sulfated proteins. We have developed a novel anti-sulfotyrosine monoclonal antibody, called PSG2, that binds with high affinity and exquisite specificity to sulfotyrosine residues in peptides and proteins independent of sequence context. We show that it can detect tyrosinesulfated proteins in complex biological samples and can be used as a probe to assess the role of tyrosine sulfation in protein function. We also demonstrate the utility of PSG2 in the purification of tyrosine-sulfated proteins from crude tissue samples. Finally, Western blot analysis using PSG2 indicates that certain sperm/epididymal proteins are undersulfated in Tpst2−/− mice. This indicates that TPST-1 and TPST-2 have distinct macromolecular substrate specificities and provides clues as to the molecular mechanism of the infertility of Tpst2−/− males. PSG2 should be widely applicable for identification of tyrosine-sulfated proteins in other systems and organisms. PMID:17046811

Tyrosine kinase inhibitors induced immune thrombocytopenia in chronic myeloid leukemia?

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Avital F. Barak

2011-12-01

Full Text Available The outcome and quality of life of chronic myeloid leukemia (CML patients has remarkably changed with the treatment of tyrosine kinase inhibitors (TKIs. Currently, hematopoietic stem cell transplantation (HSCT is considered mainly as a third line salvage therapy in cases of TKIs resistance or intolerance. Here we describe a patient with chronic phase CML who developed both resistance and late occurrence of s severe thrombocytopenia on first and second generation TKIs and eventually underwent HSCT. Although the mechanism of the myelosuppression is not fully understood, we showed for the first time the development of dose dependent platelet antibodies in the presence of TKIs, suggesting the possibility of TKIs induced thrombocytopenia. Our case emphasizes that late development of severe myelosuppression during imatinib treatment is probably an important indication for consideration of early HSCT.

Early pregnancy angiogenic markers and spontaneous abortion

DEFF Research Database (Denmark)

Andersen, Louise B; Dechend, Ralf; Karumanchi, S Ananth

2016-01-01

BACKGROUND: Spontaneous abortion is the most commonly observed adverse pregnancy outcome. The angiogenic factors soluble Fms-like kinase 1 and placental growth factor are critical for normal pregnancy and may be associated to spontaneous abortion. OBJECTIVE: We investigated the association between...... maternal serum concentrations of soluble Fms-like kinase 1 and placental growth factor, and subsequent spontaneous abortion. STUDY DESIGN: In the prospective observational Odense Child Cohort, 1676 pregnant women donated serum in early pregnancy, gestational week ..., interquartile range 71-103). Concentrations of soluble Fms-like kinase 1 and placental growth factor were determined with novel automated assays. Spontaneous abortion was defined as complete or incomplete spontaneous abortion, missed abortion, or blighted ovum

Icotinib (BPI-2009H), a novel EGFR tyrosine kinase inhibitor, displays potent efficacy in preclinical studies.

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Tan, Fenlai; Shen, Xiaoyan; Wang, Dechang; Xie, Guojian; Zhang, Xiaodong; Ding, Lieming; Hu, Yunyan; He, Wei; Wang, Yanping; Wang, Yinxiang

2012-05-01

Icotinib, one of the leading compounds selected from our compound library, was found to be a potent and specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) with an IC(50) of 5 nM. When profiled with 88 kinases, Icotinib only showed meaningful inhibitory activity to EGFR and its mutants. Icotinib blocked EGFR-mediated intracellular tyrosine phosphorylation (IC(50)=45 nM) in the human epidermoid carcinoma A431 cell line and inhibits tumor cell proliferation. In vivo studies demonstrated that Icotinib exhibited potent dose-dependent antitumor effects in nude mice carrying a variety of human tumor-derived xenografts. The drug was well tolerated at doses up to 120 mg/kg/day in mice without mortality or significant body weight loss during the treatment. A head to head randomized, double blind phase III trial using Gefitinib as an active control for patients with advanced non-small cell lung cancer (NSCLC) was finished recently (Trial registration ID: NCT01040780). The data shows that Icotinib was non-inferior to Gefitinib in terms of median progression free survival (PFS) and safety superior favor to Icotinib compared to Gefitinib. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

A bipolar clamp mechanism for activation of Jak-family protein tyrosine kinases.

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Dipak Barua

2009-04-01

Full Text Available Most cell surface receptors for growth factors and cytokines dimerize in order to mediate signal transduction. For many such receptors, the Janus kinase (Jak family of non-receptor protein tyrosine kinases are recruited in pairs and juxtaposed by dimerized receptor complexes in order to activate one another by trans-phosphorylation. An alternative mechanism for Jak trans-phosphorylation has been proposed in which the phosphorylated kinase interacts with the Src homology 2 (SH2 domain of SH2-B, a unique adaptor protein with the capacity to homo-dimerize. Building on a rule-based kinetic modeling approach that considers the concerted nature and combinatorial complexity of modular protein domain interactions, we examine these mechanisms in detail, focusing on the growth hormone (GH receptor/Jak2/SH2-Bbeta system. The modeling results suggest that, whereas Jak2-(SH2-Bbeta(2-Jak2 heterotetramers are scarcely expected to affect Jak2 phosphorylation, SH2-Bbeta and dimerized receptors synergistically promote Jak2 trans-activation in the context of intracellular signaling. Analysis of the results revealed a unique mechanism whereby SH2-B and receptor dimers constitute a bipolar 'clamp' that stabilizes the active configuration of two Jak2 molecules in the same macro-complex.

[Literature review and presentation of our own research results regarding the effects on bone of tyrosine kinase inhibitors imatinib and nilotinib used in the treatment of oncohematological diseases].

Science.gov (United States)

Kirschner, Gyöngyi; Balla, Bernadett; Kósa, János; Horváth, Péter; Kövesdi, Andrea; Lakatos, Gergely; Takács, István; Nagy, Zsolt; Tóbiás, Bálint; Árvai, Kristóf; Lakatos, Péter

2016-09-01

Tyrosine kinase inhibitors are widely used for treatment of certain oncohematological diseases. Several clinical studies have confirmed that specific BCR-ABL tyrosine kinase inhibitors alter the physiological process of bone tissue in a complex and unclearly identified manner. Since these treatments are being given to more and more patients, and the therapy takes decades or lasts even lifelong, it is justifiable to obtain more detailed knowledge of the molecular background of these mechanisms. In this article the authors summarize preliminary research results and human clinical observations on imatinib and nilotinib which are related to bone metabolism, and present the results of their own experiments in in vitro osteoblast cultures. Based on the presented results, the effects of imatinib and nilotinib on bone cells depend on the concentration of imatinib and nilotinib, the maturation stage of the cells and the distribution ratio of receptor tyrosine kinase signaling pathways. In this study the authors firstly prepared a stop-gap, comprehensive review in the Hungarian literature, regarding the effects of tyrosine kinase inhibitors on bone metabolism. In addition they firstly performed whole transcriptome analysis on osteoblasts in order to obtain a better understanding of the cellular molecular mechanisms. Orv. Hetil., 2016, 157(36), 1429-1437.

Development of a PET tracer for imaging EGFR tyrosine kinase: evaluation of the suitability of PKI166

International Nuclear Information System (INIS)

Kernchen, R.; Brust, P.; Krause, M.; Baumann, M.

2002-01-01

The suitability of PKI166 for the development of a PET tracer for imaging EGFR tyrosine kinase was investigated. Binding studies using EGFR positive tumour tissue and tritiated PKI166 as the radioligand indicated a low binding affinity of PKI166 to the target tissue. PKI166 is therefore not recommended for PET tracer development. (orig.)

Ibrutinib (PCI-32765), the first BTK (Bruton's tyrosine kinase) inhibitor in clinical trials.

Science.gov (United States)

Brown, Jennifer R

2013-03-01

Ibrutinib is a potent covalent kinase inhibitor that targets BTK. BTK, or Bruton's tyrosine kinase, is an obvious target for therapy of B cell diseases because inactivating mutations lead to B cell aplasia in humans and the disease X-linked agammaglobulinemia. Ibrutinib has modest cytotoxicity against CLL cells in vitro but also blocks trophic stimuli from the microenvironment. As with other inhibitors of the BCR pathway, ibrutinib causes rapid nodal reduction and response associated with rapid increase in lymphocytosis, which then returns to baseline over time. The ORR of ibrutinib in relapsed refractory CLL is 67 % with PFS 88 % at 15 months. In a cohort of untreated patients 65 years and over, the estimated 15 month PFS is 96 %. Registration trials have been initiated, and the difficult task that remains is to determine where in the course of CLL therapy this drug will have the greatest impact and benefit for patients.

Prolonged hypoxic culture and trypsinization increase the pro-angiogenic potential of human adipose tissue-derived stem cells

DEFF Research Database (Denmark)

Rasmussen, Jeppe Grøndahl; Frøbert, Ole; Pilgaard, Linda

2011-01-01

Transplantation of mesenchymal stromal cells (MSC), including adipose tissue-derived stem cells (ASC), is a promising option in the treatment of vascular disease. Short-term hypoxic culture of MSC augments secretion of anti-apoptotic and angiogenic cytokines. We hypothesized that prolonged hypoxic...... (1% and 5% oxygen) culture and trypsinization would augment ASC expression of anti-apoptotic and angiogenic cytokines and increase the angiogenic potential of ASC-conditioned media....

K20E, an oxidative-coupling compound of methyl caffeate, exhibits anti-angiogenic activities through down-regulations of VEGF and VEGF receptor-2

Energy Technology Data Exchange (ETDEWEB)

Pan, Chun-Hsu [Department of Pharmacy, Taipei Medical University, Taipei 11031, Taiwan (China); Lin, Wen-Hsin; Chien, Yi-Chung; Liu, Fon-Chang; Sheu, Ming-Jyh [School of Pharmacy, China Medical University, Taichung 40402, Taiwan (China); Kuo, Yueh-Hsiung, E-mail: kuoyh@mail.cmu.edu.tw [Tsuzuki Institute for Traditional Medicine, China Medical University, Taichung 40402, Taiwan (China); Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung 40402, Taiwan (China); Department of Biotechnology, Asia University, Taichung 41354, Taiwan (China); Wu, Chieh-Hsi, E-mail: chhswu@tmu.edu.tw [Department of Pharmacy, Taipei Medical University, Taipei 11031, Taiwan (China); School of Pharmacy, China Medical University, Taichung 40402, Taiwan (China); Department of Biological Science and Technology, China Medical University, Taichung 40402, Taiwan (China)

2015-01-15

Anti-angiogenesis is one of the most popular clinical interventions for cancer chemotherapy. A series of synthesized derivative of methyl caffeate were used to evaluate the anti-angiogenic activity and to investigate possible pharmacological mechanisms in the present study. The most potent anti-angiogenic compound was evaluated in the experiments of murine allograft tumor model and Matrigel plug assay as well as cell models in the human umbilical vascular endothelial cells (HUVECs) and the LLC1 lung cancer cells. Our results suggested that K20E suppressed the tumor growth in the allograft tumor model and exhibited anti-angiogenic activity in Matrigel plug assay. Besides, HUVEC viability was found to be significantly reduced by arresting cell cycle at G{sub 2}/M phase and apoptosis. Cell migration, invasion, and tube formation of the HUVECs were also markedly suppressed by K20E treatment. K20E largely down-regulated the intracellular and secreted vascular endothelial growth factor (VEGF) in the LLC1 cancer cells. Besides, VEGF receptor-2 (VEGFR-2) and its downstream signaling cascades (AKT-mTOR and MEK1/2-ERK1/2) as well as gelatinases were all evidently reduced in the HUVECs treated with K20E. Inversely, K20E can up-regulate the expression levels of p53 and p21 proteins in the HUVECs. Based on these results, our study suggested that K20E possessed inhibiting angiogenesis through regulation of VEGF/VEGFR-2 and its downstream signaling cascades in the vascular endothelial cells (VECs). - Highlights: • K20E is an oxidative-coupling compound of methyl caffeate. • K20E exhibits anti-tumor and anti-angiogenesis effects. • K20E suppresses the expressions of VEGF and VEGF receptor-2 (VEGFR-2) proteins. • K20E deactivates VEGFR-2-mediated downstream signaling pathways to inhibit angiogenesis. • K20E up-regulates p53-p21 pathway to induce apoptosis and cell arrest at G2/M phase.

Structural coupling of SH2-kinase domains links Fes and Abl substrate recognition and kinase activation.

Science.gov (United States)

Filippakopoulos, Panagis; Kofler, Michael; Hantschel, Oliver; Gish, Gerald D; Grebien, Florian; Salah, Eidarus; Neudecker, Philipp; Kay, Lewis E; Turk, Benjamin E; Superti-Furga, Giulio; Pawson, Tony; Knapp, Stefan

2008-09-05

The SH2 domain of cytoplasmic tyrosine kinases can enhance catalytic activity and substrate recognition, but the molecular mechanisms by which this is achieved are poorly understood. We have solved the structure of the prototypic SH2-kinase unit of the human Fes tyrosine kinase, which appears specialized for positive signaling. In its active conformation, the SH2 domain tightly interacts with the kinase N-terminal lobe and positions the kinase alphaC helix in an active configuration through essential packing and electrostatic interactions. This interaction is stabilized by ligand binding to the SH2 domain. Our data indicate that Fes kinase activation is closely coupled to substrate recognition through cooperative SH2-kinase-substrate interactions. Similarly, we find that the SH2 domain of the active Abl kinase stimulates catalytic activity and substrate phosphorylation through a distinct SH2-kinase interface. Thus, the SH2 and catalytic domains of active Fes and Abl pro-oncogenic kinases form integrated structures essential for effective tyrosine kinase signaling.

Response to Plasmapheresis Measured by Angiogenic Factors in a Woman with Antiphospholipid Syndrome in Pregnancy

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Karoline Mayer-Pickel

2015-01-01

Full Text Available An imbalance of angiogenic and antiangiogenic placental factors such as endoglin and soluble fms-like tyrosine kinase 1 has been implicated in the pathophysiology of preeclampsia. Extraction of these substances by plasmapheresis might be a therapeutical approach in cases of severe early-onset preeclampsia. Case Report. A 21-year-old primigravida with antiphospholipid syndrome developed early-onset preeclampsia at 18 weeks’ gestation. She was treated successfully with plasmapheresis in order to prolong pregnancy. Endoglin and sflt-1-levels were measured by ELISA before and after treatment. Endoglin levels decreased significantly after treatment (p < 0.05 and showed a significant decrease throughout pregnancy. A rerise of endoglin and sflt-1 preceded placental abruption 4 weeks before onset of incident. Conclusion. Due to the limited long-term therapeutical possibilities for pregnancies complicated by PE, plasmapheresis seems to be a therapeutical option. This consideration refers especially to pregnancies with early-onset preeclampsia, in which, after first conventional treatment of PE, prolongation of pregnancy should be above all.

2D QSAR studies of the inhibitory activity of a series of substituted purine derivatives against c-Src tyrosine kinase

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Mukesh C. Sharma

2016-07-01

Full Text Available A series of 34 substituted purine analogues derivatives were subjected to quantitative structure-activity relationship analyses as inhibitors of c-Src tyrosine kinase. Partial least squares regression was applied to derive QSAR models, which were further validated for statistical significance by internal and external validation. The best QSAR model developed had a good predictive correlation coefficient (r2 of 0.8319, a significant cross-validated correlation coefficient (q2 of 0.7550, and an r2 for the external test set (pred_r2 of 0.7983. It was developed from the PLS method with descriptors including the SsCH3E-index, H-Donor Count, T_2_Cl_3, and negative correlation with SsOHcount. The current study provides better insight into the future design of more potent c-Src tyrosine kinase inhibitors prior to synthesis.

Human chorionic gonadotropin, angiogenic factors, and preeclampsia risk: a nested case-control study.

Science.gov (United States)

Asvold, Bjørn O; Eskild, Anne; Vatten, Lars J

2014-05-01

To study whether human chorionic gonadotropin concentrations during pregnancy or combinations of human chorionic gonadotropin and other angiogenic factors, soluble fms-like tyrosine kinase 1 and placental growth factor (PlGF), are associated with preeclampsia risk. Nested case-control study. Population cohort of pregnant women. A total of 121 cases of preterm (cases of term preeclampsia (≥37 weeks of gestation) and 356 women without preeclampsia (controls). Women with preeclampsia were identified by linkage to the Medical Birth Registry of Norway. Concentrations of human chorionic gonadotropin, soluble fms-like tyrosine kinase 1 and PlGF were measured in maternal serum samples collected in each trimester of pregnancy. Odds ratios of preterm and term preeclampsia. High human chorionic gonadotropin concentrations (highest quartile) in the first trimester were associated with reduced risk for preterm preeclampsia (OR 0.3, 95% CI 0.1-0.9), compared with low human chorionic gonadotropin (lowest quartile), whereas high human chorionic gonadotropin concentrations in the second trimester were associated with increased risk for preterm preeclampsia (OR 4.0, 95% CI 1.8-8.9). High human chorionic gonadotropin concentrations in the third trimester were associated with increased risk for term preeclampsia (OR 4.8, 95% CI 1.8-13.3). Concentrations of human chorionic gonadotropin above the median value combined with PlGF below the median in the second trimester were associated with very high risk for preterm preeclampsia (OR 36.9, 95% CI 8.2-165.8). The results suggest an important role of human chorionic gonadotropin in the pathophysiological processes that lead to preeclampsia. The combined association of human chorionic gonadotropin and PlGF indicates a possible synergism between underlying biological pathways. © 2014 Nordic Federation of Societies of Obstetrics and Gynecology.

Sesquiterpene dimmer (DSF-27) inhibits the release of neuroinflammatory mediators from microglia by targeting spleen tyrosine kinase (Syk) and Janus kinase 2 (Jak2): Two major non-receptor tyrosine signaling proteins involved in inflammatory events

Energy Technology Data Exchange (ETDEWEB)

Zeng, Ke-Wu [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China); Wang, Shu [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China); Department of Medicinal Chemistry and Pharmaceutical Analysis, Logistics College of Chinese People' s Armed Police Forces, Tianjin 300162 (China); Dong, Xin; Jiang, Yong; Jin, Hong-Wei [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China); Tu, Peng-Fei, E-mail: pengfeitu@vip.163.com [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China)

2014-03-15

Non-receptor protein tyrosine kinases (NRPTKs)-dependent inflammatory signal transduction cascades play key roles in immunoregulation. However, drug intervention through NRPTKs-involved immunoregulation mechanism in microglia (the major immune cells of the central nervous system) has not been widely investigated. A main aim of the present study is to elucidate the contribution of two major NRPTKs (Syk and Jak2) in neuroinflammation suppression by a bioactive sesquiterpene dimmer (DSF-27). We found that LPS-stimulated BV-2 cells activated Syk and further initiated Akt/NF-κB inflammatory pathway. This Syk-dependent Akt/NF-κB inflammatory pathway can be effectively ameliorated by DSF-27. Moreover, Jak2 was activated by LPS, which was followed by transcriptional factor Stat3 activation. The Jak2/Stat3 signal was suppressed by DSF-27 through inhibition of Jak2 and Stat3 phosphorylation, promotion of Jak/Stat3 inhibitory factors PIAS3 expression, and down-regulation of ERK and p38 MAPK phosphorylation. Furthermore, DSF-27 protected cortical and mesencephalic dopaminergic neurons against neuroinflammatory injury. Taken together, our findings indicate NRPTK signaling pathways including Syk/NF-κB and Jak2/Stat3 cascades are potential anti-neuroinflammatory targets in microglia, and may also set the basis for the use of sesquiterpene dimmer as a therapeutic approach for neuroinflammation via interruption of these pathways. - Highlights: • Sesquiterpene dimmer DSF-27 inhibits inflammatory mediators' production in microglia. • Syk-dependent Akt/NF-κB pathway is important for DSF-27's anti-inflammation activity. • Jak2/Stat3 pathway is important for DSF-27's anti-inflammation activity. • Jak2/Stat3 signaling pathway is partly regulated by ERK and p38 MAPKs and PIAS3. • DSF-27 protects neurons against microglia-mediated neuroinflammatory injury.

MHC class I ligation of human T cells activates the ZAP70 and p56lck tyrosine kinases, leads to an alternative phenotype of the TCR/CD3 zeta-chain, and induces apoptosis

DEFF Research Database (Denmark)

Skov, S; Bregenholt, S; Claesson, Mogens Helweg

1997-01-01

Cross-linking of MHC class I (MHC-I) molecules on human T cells induces signal-transduction events, including activation of tyrosine kinases, tyrosine phosphorylation of phospholipase C-gamma 1, and elevation of the intracellular free calcium concentration. In this study, we demonstrate...... that the ZAP70 tyrosine kinase is tyrosine phosphorylated in Jurkat T cells and in purified peripheral T cells after MHC-I ligation. The tyrosine-phosphorylated ZAP70 kinase exhibits a particular phenotype with low affinities for proteins at 21, 40, 60, and 120 kDa, proteins normally co-precipitated with ZAP70...... after TCR/CD3 stimulation. The phosphorylation of ZAP70 after MHC-I ligation was dependent on TCR/CD3 surface expression. One of the natural substrates for ZAP70 is the zeta-chain dimer of the TCR/CD3 complex. MHC-I cross-linking induces a phosphorylated zeta-protein that migrates as a dimer at 42 k...

Interleukins 2, 4, 7, and 15 stimulate tyrosine phosphorylation of insulin receptor substrates 1 and 2 in T cells. Potential role of JAK kinases.

Science.gov (United States)

Johnston, J A; Wang, L M; Hanson, E P; Sun, X J; White, M F; Oakes, S A; Pierce, J H; O'Shea, J J

1995-12-01

The signaling molecules insulin receptor substrate (IRS)-1 and the newly described IRS-2 (4PS) molecule are major insulin and interleukin 4 (IL-4)-dependent phosphoproteins. We report here that IL-2, IL-7, and IL-15, as well as IL-4, rapidly stimulate the tyrosine phosphorylation of IRS-1 and IRS-2 in human peripheral blood T cells, NK cells, and in lymphoid cell lines. In addition, we show that the Janus kinases, JAK1 and JAK3, associate with IRS-1 and IRS-2 in T cells. Coexpression studies demonstrate that these kinases can tyrosine-phosphorylate IRS-2, suggesting a possible mechanism by which cytokine receptors may induce the tyrosine phosphorylation of IRS-1 and IRS-2. We further demonstrate that the p85 subunit of phosphoinositol 3-kinase associates with IRS-1 in response to IL-2 and IL-4 in T cells. Therefore, these data indicate that IRS-1 and IRS-2 may have important roles in T lymphocyte activation not only in response to IL-4, but also in response to IL-2, IL-7, and IL-15.

syk kinase activation by a src kinase-initiated activation loop phosphorylation chain reaction

Science.gov (United States)

El-Hillal, O.; Kurosaki, T.; Yamamura, H.; Kinet, J.-P.; Scharenberg, A. M.

1997-01-01

Activation of the syk tyrosine kinase occurs almost immediately following engagement of many types of antigen receptors, including Fc receptors, but the mechanism through which syk is activated is currently unclear. Here we demonstrate that Fc receptor-induced syk activation occurs as the result of phosphorylation of the syk activation loop by both src family kinases and other molecules of activated syk, suggesting that syk activation occurs as the result of a src kinase-initiated activation loop phosphorylation chain reaction. This type of activation mechanism predicts that syk activation would exhibit exponential kinetics, providing a potential explanation for its rapid and robust activation by even weak antigen receptor stimuli. We propose that a similar mechanism may be responsible for generating rapid activation of other cytoplasmic tyrosine kinases, such as those of the Bruton tyrosine kinase/tec family, as well. PMID:9050880

Tyrosine phosphorylation in T cells is regulated by phosphatase activity: studies with phenylarsine oxide.

OpenAIRE

Garcia-Morales, P; Minami, Y; Luong, E; Klausner, R D; Samelson, L E

1990-01-01

Activation of T cells induces rapid tyrosine phosphorylation on the T-cell receptor zeta chain and other substrates. These phosphorylations can be regulated by a number of protein-tyrosine kinases (ATP: protein-tyrosine O-phosphotransferase, EC 2.7.1.112) and protein-tyrosine-phosphatases (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48). In this study, we demonstrate that phenylarsine oxide can inhibit tyrosine phosphatases while leaving tyrosine kinase function intact. We use this ...

Embolization biomaterial reinforced with nanotechnology for an in-situ release of anti-angiogenic agent in the treatment of hyper-vascularized tumors and arteriovenous malformations.

Science.gov (United States)

Jubeli, E; Yagoubi, N; Pascale, F; Bédouet, L; Slimani, K; Labarre, D; Saint-Maurice, J P; Laurent, A; Moine, L

2015-10-01

A polymer based material was developed to act as an embolic agent and drug reservoir for the treatment of arteriovenous malformations (AVM) and hyper vascularized solid tumors. The aim was to combine the blocking of blood supply to the target region and the inhibition of the embolization-stimulated angiogenesis. The material is composed of an ethanolic solution of a linear acrylate based copolymer and acrylate calibrated microparticles containing nanospheres loaded with sunitinib, an anti-angiogenic agent. The precipitation of the linear copolymer in aqueous environment after injection through microcatheter results in the formation of an in-situ embolization gel whereas the microparticles serve to increase the cohesive properties of the embolization agent and to form a reservoir from which the sunitinib-loaded nanospheres are released post-embolization. The swollen state of the microparticles in contact with aqueous medium results in the release of the nanospheres out of microparticles macromolecular structure. After the synthesis, the formulation and the characterization of the different components of the material, anti-angiogenic activity was evaluated in vitro using endothelial cells and in vivo using corneal neovascularization model in rabbit. The efficiency of the arterial embolization was tested in vivo in a sheep model. Results proved the feasibility of this new system for vascular embolization in association with an in situ delivery of anti-angiogenic drug. This combination is a promising strategy for the management of arteriovenous malformations and solid tumors. Copyright © 2015 Elsevier B.V. All rights reserved.

The anti-esophageal cancer cell activity by a novel tyrosine/phosphoinositide kinase inhibitor PP121

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Peng, Yi; Zhou, Yajuan [Department of Radiation and Medical Oncology, Hubei Key Laboratory of Tumor Biological Behaviors, Zhongnan Hospital of Wuhan University, Wuhan 430071 (China); Department of Radiation Oncology, Hubei Cancer Hospital, Wuhan 430071 (China); Cheng, Long [Department of Interventional Radiology, the Second Affiliated Hospital of Soochow University, Soochow University, Suzhou 215001 (China); Hu, Desheng; Zhou, Xiaoyi; Wang, Zhaohua [Department of Radiation Oncology, Hubei Cancer Hospital, Wuhan 430071 (China); Xie, Conghua, E-mail: chxie_65@hotmail.com [Department of Radiation and Medical Oncology, Hubei Key Laboratory of Tumor Biological Behaviors, Zhongnan Hospital of Wuhan University, Wuhan 430071 (China); Zhou, Fuxiang, E-mail: ZhouFuxiangwuhan@126.com [Department of Radiation and Medical Oncology, Hubei Key Laboratory of Tumor Biological Behaviors, Zhongnan Hospital of Wuhan University, Wuhan 430071 (China)

2015-09-11

Here we explored the potential effect of PP121, a novel dual inhibitor of tyrosine and phosphoinositide kinases, against human esophageal cancer cells. We showed that PP121 exerted potent cytotoxic effect in primary (patient-derived) and established (Eca-109, TE-1 and TE-3 lines) esophageal cancer cells, possibly through activating caspase-3-dependnent apoptosis. PP121 was, however, non-cytotoxic to the normal human esophageal epithelial cells (EECs). At the molecular level, we showed that PP121 blocked Akt-mTOR (mammalian target of rapamycin) activation in esophageal cancer cells, which was restored by introducing a constitutively-active Akt (CA-Akt). Yet, CA-Akt only partly inhibited cytotoxicity by PP121 in Eca-109 cells. Importantly, we showed that PP121 inhibited nuclear factor kappa B (NFκB) signaling activation in esophageal cancer cells, which appeared independent of Akt-mTOR blockage. In vivo, oral administration of PP121 remarkably inhibited Eca-109 xenograft growth in nude mice, and significantly improved mice survival. Further, the immunohistochemistry (IHC) and Western blot assays analyzing xenografted tumors showed that PP121 inhibited Akt-mTOR and NFκB activations in vivo. Together, we demonstrate that PP121 potently inhibits esophageal cancer cells in vitro and in vivo, possibly through concurrently inhibiting Akt-mTOR and NFκB signalings. - Highlights: • PP121 is cytotoxic against primary and established esophageal cancer cells. • PP121 induces caspase-3-dependnent apoptosis in esophageal cancer cells. • PP121 blocks Akt-mTOR activation in esophageal cancer cells. • PP121 inhibits NFκB activation, independent of Akt-mTOR blockage. • PP121 inhibits Eca-109 xenograft growth and Akt-mTOR/NFκB activation in vivo.

Nuclear localization of lymphocyte-specific protein tyrosine kinase (Lck) and its role in regulating LIM domain only 2 (Lmo2) gene

Energy Technology Data Exchange (ETDEWEB)

Venkitachalam, Srividya; Chueh, Fu-Yu [Department of Microbiology and Immunology, H. M. Bligh Cancer Research Laboratories, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064 (United States); Yu, Chao-Lan, E-mail: chaolan.yu@rosalindfranklin.edu [Department of Microbiology and Immunology, H. M. Bligh Cancer Research Laboratories, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064 (United States)

2012-01-20

Highlights: Black-Right-Pointing-Pointer Lmo2 expression is elevated in Lck-transformed cells. Black-Right-Pointing-Pointer Both endogenous and exogenous Lck localize in the nucleus. Black-Right-Pointing-Pointer Nuclear Lck is active in Lck-transformed cells. Black-Right-Pointing-Pointer Lck binds to the promoter region of Lmo2 gene in vivo. Black-Right-Pointing-Pointer In contrast to JAK2, Lck does not increase histone H3 phosphorylation on Tyr 41. -- Abstract: LIM domain only protein 2 (Lmo2) is a transcription factor that plays a critical role in the development of T-acute lymphoblastic leukemia (T-ALL). A previous report established a link between Lmo2 expression and the nuclear presence of oncogenic Janus kinase 2 (JAK2), a non-receptor protein tyrosine kinase. The oncogenic JAK2 kinase phosphorylates histone H3 on Tyr 41 that leads to the relief of Lmo2 promoter repression and subsequent gene expression. Similar to JAK2, constitutive activation of lymphocyte-specific protein tyrosine kinase (Lck) has been implicated in lymphoid malignancies. However, it is not known whether oncogenic Lck regulates Lmo2 expression through a similar mechanism. We show here that Lmo2 expression is significantly elevated in T cell leukemia LSTRA overexpressing active Lck kinase and in HEK 293 cells expressing oncogenic Y505FLck kinase. Nuclear localization of active Lck kinase was confirmed in both Lck-transformed cells by subcellular fractionation and immunofluorescence microscopy. More importantly, in contrast to oncogenic JAK2, oncogenic Lck kinase does not result in significant increase in histone H3 phosphorylation on Tyr 41. Instead, chromatin immunoprecipitation experiment shows that oncogenic Y505FLck kinase binds to the Lmo2 promoter in vivo. This result raises the possibility that oncogenic Lck may activate Lmo2 promoter through direct interaction.

Platelet-activating factor stimulation of tyrosine kinase and its relationship to phospholipase C in rabbit platelets: Studies with genistein and monoclonal antibody to phosphotyrosine

International Nuclear Information System (INIS)

Dhar, A.; Paul, A.K.; Shukla, S.D.

1990-01-01

Platelet-activating factor (PAF) is a proinflammatory lipid that has platelet-stimulating property. PAF receptor-coupled activation of phosphoinositide-specific phospholipase C (PLC) and phosphorylation of several proteins has already been established in our laboratory. To investigate further the molecular mechanism and relationship between activation of PLC and protein phosphorylation, we have used Genistein (a putative inhibitor of tyrosine-specific protein kinases), phosphotyrosine antibody, and phosphoamino acid analysis to probe the involvement of tyrosine kinase in this process. Washed rabbit platelets were loaded with myo-[2-3H]inositol and challenged with PAF (100 nM) after pretreatment with Genistein. PLC-mediated production of radioactive inositol monophosphate, inositol diphosphate, and inositol triphosphate was monitored. PAF alone caused stimulation of PLC activity [( 3H]inositol triphosphate production), whereas pretreatment with Genistein (0.5 mM) diminished PAF-stimulated PLC activity to basal level. Genistein also blocked PAF-stimulated platelet aggregation at this dose. In contrast to Genistein, staurosporine which inhibits protein kinase C, potentiated PAF-stimulated [3H]inositol triphosphate production. Genistein substantially inhibited the combined effects of staurosporine and PAF on inositol triphosphate production. Genistein also reduced PAF-induced phosphorylation of Mr 20,000 and 50,000 proteins. Phorbol 12-myristate 13-acetate-induced Mr 40,000 protein phosphorylation was also affected by Genistein. The above results suggested that Genistein inhibited tyrosine kinase at an early stage of signal transduction by inhibiting PLC. This, in turn, decreased the activation of protein kinase C and, therefore, caused a reduction in Mr 40,000 protein phosphorylation

Activated Fps/Fes tyrosine kinase regulates erythroid differentiation and survival.

Science.gov (United States)

Sangrar, Waheed; Gao, Yan; Bates, Barbara; Zirngibl, Ralph; Greer, Peter A

2004-10-01

A substantial body of evidence implicates the cytoplasmic protein tyrosine kinase Fps/Fes in regulation of myeloid differentiation and survival. In this study we wished to determine if Fps/Fes also plays a role in the regulation of erythropoiesis. Mice tissue-specifically expressing a "gain-of-function" mutant fps/fes transgene (fps(MF)) encoding an activated variant of Fps/Fes (MFps), were used to explore the in vivo biological role of Fps/Fes. Erythropoiesis in these mice was assessed by hematological analysis, lineage marker analysis, bone-marrow colony assays, and biochemical approaches. fps(MF) mice displayed reductions in peripheral red cell counts. However, there was an accumulation of immature erythroid precursors, which displayed increased survival. Fps/Fes and the related Fer kinase were both detected in early erythroid progenitors/blasts and in mature red cells. Fps/Fes was also activated in response to erythropoietin (EPO) and stem cell factor (SCF), two critical factors in erythroid development. In addition, increased Stat5A/B activation and reduced Erk1/2 phosphorylation was observed in fps(MF) primary erythroid cells in response to EPO or SCF, respectively. These data support a role for Fps/Fes in regulating the survival and differentiation of erythroid cells through modulation of Stat5A/B and Erk kinase pathways induced by EPO and SCF. The increased numbers and survival of erythroid progenitors from fps(MF) mice, and their differential responsiveness to SCF and EPO, implicates Fps/Fes in the commitment of multilineage progenitors to the erythroid lineage. The anemic phenotype in fps(MF) mice suggests that downregulation of Fps/Fes activity might be required for terminal erythroid differentiation.

A protein-binding domain, EH, identified in the receptor tyrosine kinase substrate Eps15 and conserved in evolution

DEFF Research Database (Denmark)

Wong, W T; Schumacher, C; Salcini, A E

1995-01-01

In this report we structurally and functionally define a binding domain that is involved in protein association and that we have designated EH (for Eps15 homology domain). This domain was identified in the tyrosine kinase substrate Eps15 on the basis of regional conservation with several heteroge......In this report we structurally and functionally define a binding domain that is involved in protein association and that we have designated EH (for Eps15 homology domain). This domain was identified in the tyrosine kinase substrate Eps15 on the basis of regional conservation with several...... heterogeneous proteins of yeast and nematode. The EH domain spans about 70 amino acids and shows approximately 60% overall amino acid conservation. We demonstrated the ability of the EH domain to specifically bind cytosolic proteins in normal and malignant cells of mesenchymal, epithelial, and hematopoietic...... (for Eps15-related). Structural comparison of Eps15 and Eps15r defines a family of signal transducers possessing extensive networking abilities including EH-mediated binding and association with Src homology 3-containing proteins....

An in silico high-throughput screen identifies potential selective inhibitors for the non-receptor tyrosine kinase Pyk2

Directory of Open Access Journals (Sweden)

Meirson T

2017-05-01

Full Text Available Tomer Meirson, Abraham O Samson, Hava Gil-Henn Faculty of Medicine in the Galilee, Bar-Ilan University, Safed, Israel Abstract: The non-receptor tyrosine kinase proline-rich tyrosine kinase 2 (Pyk2 is a critical mediator of signaling from cell surface growth factor and adhesion receptors to cell migration, proliferation, and survival. Emerging evidence indicates that signaling by Pyk2 regulates hematopoietic cell response, bone density, neuronal degeneration, angiogenesis, and cancer. These physiological and pathological roles of Pyk2 warrant it as a valuable therapeutic target for invasive cancers, osteoporosis, Alzheimer’s disease, and inflammatory cellular response. Despite its potential as a therapeutic target, no potent and selective inhibitor of Pyk2 is available at present. As a first step toward discovering specific potential inhibitors of Pyk2, we used an in silico high-throughput screening approach. A virtual library of six million lead-like compounds was docked against four different high-resolution Pyk2 kinase domain crystal structures and further selected for predicted potency and ligand efficiency. Ligand selectivity for Pyk2 over focal adhesion kinase (FAK was evaluated by comparative docking of ligands and measurement of binding free energy so as to obtain 40 potential candidates. Finally, the structural flexibility of a subset of the docking complexes was evaluated by molecular dynamics simulation, followed by intermolecular interaction analysis. These compounds may be considered as promising leads for further development of highly selective Pyk2 inhibitors. Keywords: virtual screen, efficiency metrics, MM-GBSA, molecular dynamics

Pseudomonas aeruginosa invasion and cytotoxicity are independent events, both of which involve protein tyrosine kinase activity.

Science.gov (United States)

Evans, D J; Frank, D W; Finck-Barbançon, V; Wu, C; Fleiszig, S M

1998-04-01

Pseudomonas aeruginosa clinical isolates exhibit invasive or cytotoxic phenotypes. Cytotoxic strains acquire some of the characteristics of invasive strains when a regulatory gene, exsA, that controls the expression of several extracellular proteins, is inactivated. exsA mutants are not cytotoxic and can be detected within epithelial cells by gentamicin survival assays. The purpose of this study was to determine whether epithelial cell invasion precedes and/or is essential for cytotoxicity. This was tested by measuring invasion (gentamicin survival) and cytotoxicity (trypan blue staining) of PA103 mutants deficient in specific exsA-regulated proteins and by testing the effect of drugs that inhibit invasion for their effect on cytotoxicity. A transposon mutant in the exsA-regulated extracellular factor exoU was neither cytotoxic nor invasive. Furthermore, several of the drugs that inhibited invasion did not prevent cytotoxicity. These results show that invasion and cytotoxicity are mutually exclusive events, inversely regulated by an exsA-encoded invasion inhibitor(s). Both involve host cell protein tyrosine kinase (PTK) activity, but they differ in that invasion requires Src family tyrosine kinases and calcium-calmodulin activity. PTK inhibitor drugs such as genistein may have therapeutic potential through their ability to block both invasive and cytotoxicity pathways via an action on the host cell.

Detection of a rare BCR-ABL tyrosine kinase fusion protein in H929 multiple myeloma cells using immunoprecipitation (IP)-tandem mass spectrometry (MS/MS).

Science.gov (United States)

Breitkopf, Susanne B; Yuan, Min; Pihan, German A; Asara, John M

2012-10-02

Hypothesis directed proteomics offers higher throughput over global analyses. We show that immunoprecipitation (IP)-tandem mass spectrometry (LC-MS/MS) in H929 multiple myeloma (MM) cancer cells led to the discovery of a rare and unexpected BCR-ABL fusion, informing a therapeutic intervention using imatinib (Gleevec). BCR-ABL is the driving mutation in chronic myeloid leukemia (CML) and is uncommon to other cancers. Three different IP-MS experiments central to cell signaling pathways were sufficient to discover a BCR-ABL fusion in H929 cells: phosphotyrosine (pY) peptide IP, p85 regulatory subunit of phosphoinositide-3-kinase (PI3K) IP, and the GRB2 adaptor IP. The pY peptides inform tyrosine kinase activity, p85 IP informs the activating adaptors and receptor tyrosine kinases (RTKs) involved in AKT activation and GRB2 IP identifies RTKs and adaptors leading to ERK activation. Integration of the bait-prey data from the three separate experiments identified the BCR-ABL protein complex, which was confirmed by biochemistry, cytogenetic methods, and DNA sequencing revealed the e14a2 fusion transcript. The tyrosine phosphatase SHP2 and the GAB2 adaptor protein, important for MAPK signaling, were common to all three IP-MS experiments. The comparative treatment of tyrosine kinase inhibitor (TKI) drugs revealed only imatinib, the standard of care in CML, was inhibitory to BCR-ABL leading to down-regulation of pERK and pS6K and inhibiting cell proliferation. These data suggest a model for directed proteomics from patient tumor samples for selecting the appropriate TKI drug(s) based on IP and LC-MS/MS. The data also suggest that MM patients, in addition to CML patients, may benefit from BCR-ABL diagnostic screening.

Cord Blood Angiogenic Profile in Normotensive Pregnancies

African Journals Online (AJOL)

2017-06-30

Jun 30, 2017 ... favorable anti- to pro-angiogenic balance in pregnant women. ... tweak,and build upon the work non-commercially,as long as the author is credited and the new ..... utero blood pressure in childhood and adult life and mortality.

Lymphocyte-specific protein tyrosine kinase (Lck) interacts with CR6-interacting factor 1 (CRIF1) in mitochondria to repress oxidative phosphorylation

International Nuclear Information System (INIS)

Vahedi, Shahrooz; Chueh, Fu-Yu; Chandran, Bala; Yu, Chao-Lan

2015-01-01

Many cancer cells exhibit reduced mitochondrial respiration as part of metabolic reprogramming to support tumor growth. Mitochondrial localization of several protein tyrosine kinases is linked to this characteristic metabolic shift in solid tumors, but remains largely unknown in blood cancer. Lymphocyte-specific protein tyrosine kinase (Lck) is a key T-cell kinase and widely implicated in blood malignancies. The purpose of our study is to determine whether and how Lck contributes to metabolic shift in T-cell leukemia through mitochondrial localization. We compared the human leukemic T-cell line Jurkat with its Lck-deficient derivative Jcam cell line. Differences in mitochondrial respiration were measured by the levels of mitochondrial membrane potential, oxygen consumption, and mitochondrial superoxide. Detailed mitochondrial structure was visualized by transmission electron microscopy. Lck localization was evaluated by subcellular fractionation and confocal microscopy. Proteomic analysis was performed to identify proteins co-precipitated with Lck in leukemic T-cells. Protein interaction was validated by biochemical co-precipitation and confocal microscopy, followed by in situ proximity ligation assay microscopy to confirm close-range (<16 nm) interaction. Jurkat cells have abnormal mitochondrial structure and reduced levels of mitochondrial respiration, which is associated with the presence of mitochondrial Lck and lower levels of mitochondrion-encoded electron transport chain proteins. Proteomics identified CR6-interacting factor 1 (CRIF1) as the novel Lck-interacting protein. Lck association with CRIF1 in Jurkat mitochondria was confirmed biochemically and by microscopy, but did not lead to CRIF1 tyrosine phosphorylation. Consistent with the role of CRIF1 in functional mitoribosome, shRNA-mediated silencing of CRIF1 in Jcam resulted in mitochondrial dysfunction similar to that observed in Jurkat. Reduced interaction between CRIF1 and Tid1, another key component

Cyclic versus Hemi-Bastadins. Pleiotropic Anti-Cancer Effects: from Apoptosis to Anti-Angiogenic and Anti-Migratory Effects

Directory of Open Access Journals (Sweden)

Peter Proksch

2013-03-01

Full Text Available Bastadins-6, -9 and -16 isolated from the marine sponge Ianthella basta displayed in vitro cytostatic and/or cytotoxic effects in six human and mouse cancer cell lines. The in vitro growth inhibitory effects of these bastadins were similar in cancer cell lines sensitive to pro-apoptotic stimuli versus cancer cell lines displaying various levels of resistance to pro-apoptotic stimuli. While about ten times less toxic than the natural cyclic bastadins, the synthetically derived 5,5'-dibromohemibastadin-1 (DBHB displayed not only in vitro growth inhibitory activity in cancer cells but also anti-angiogenic properties. At a concentration of one tenth of its in vitro growth inhibitory concentration, DBHB displayed actual antimigratory effects in mouse B16F10 melanoma cells without any sign of cytotoxicity and/or growth inhibition. The serum concentration used in the cell culture media markedly influenced the DBHB-induced antimigratory effects in the B16F10 melanoma cell population. We are currently developing a specific inhalation formulation for DBHB enabling this compound to avoid plasmatic albumin binding through its direct delivery to the lungs to combat primary as well as secondary (metastases tumors.

Fps/Fes protein-tyrosine kinase regulates mast cell adhesion and migration downstream of Kit and beta1 integrin receptors.

Science.gov (United States)

Smith, Julie A; Samayawardhena, Lionel A; Craig, Andrew W B

2010-03-01

Activation of Kit receptor protein-tyrosine kinase (PTK) by its ligand Stem Cell Factor (SCF) is required for the development of mast cells, and for the regulation of mast cell proliferation, migration and modulation of inflammatory mediator release. Recent studies have implicated the non-receptor PTK Fps/Fes (hereafter referred to as Fes) in signaling downstream of oncogenic Kit, however, the potential role of Fes in regulating Kit signaling is not well defined. In this study, we show that SCF induces transient tyrosine phosphorylation of wild-type Fes as well as kinase-dead Fes in bone marrow-derived mast cells (BMMCs). The latter finding implicates an upstream kinase acting on Fes, which we identified as Fyn PTK. SCF treatment of BMMCs promoted recruitment of Fes to Kit, potentially via direct interaction of the Fes SH2 domain with phosphorylated Kit. While Fes was not required for SCF-induced signaling to Akt and Erk kinases, Fes-deficient (fes-/-) BMMCs displayed a defect in sustained p38 kinase activation, compared to control cells. SCF-treated Fes-deficient BMMCs also displayed elevated beta1 integrin-mediated cell adhesion and spreading on fibronectin, compared to control cells, and a reduction in cell polarization at later times of SCF treatment. Restoring Fes expression in fes-/- BMMCs by retroviral transduction was sufficient to rescue cell spreading and polarization defects. Interestingly, SCF-induced chemotaxis of BMMCs was also defective in Fes-deficient BMMCs, and restored in Fes-rescue BMMCs. Overall, these results implicate Fes in regulating cross-talk between Kit and beta1 integrins to promote cytoskeletal reorganization and motility of mast cells.

Liquid chromatography-tandem mass spectrometric assay for the tyrosine kinase inhibitor afatinib in mouse plasma using salting-out liquid-liquid extraction

NARCIS (Netherlands)

Sparidans, Rolf W; van Hoppe, Stephanie; Rood, Johannes J M; Schinkel, Alfred H; Schellens, Jan H M; Beijnen, Jos H

2016-01-01

A quantitative bioanalytical liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay for afatinib, an irreversible inhibitor of the ErbB (erythroblastic leukemia viral oncogene homolog) tyrosine kinase family, was developed and validated. Plasma samples were pre-treated using salting-out

Phosphorylated human prolactin (S179D-hPRL) is a potent anti-angiogenic hormone in vitro and in vivo; Prolactina humana pseudofosforilada (S179D-hPRL) e um potente fator anti-angiogenico in vitro e in vivo

Energy Technology Data Exchange (ETDEWEB)

Ueda, Eric Kinnosuke Martins

2006-07-01

previously shown, S179D-hPRL blocked extracellular regulated kinase (ERK) phosphorylation in response to bFGF, but, in addition, continued co-incubation showed a delayed and prolonged activation of ERK. PD98059 [a specific mitogen-activated protein kinase (MAPkinase) inhibitor] inhibited this delayed activation of ERK and the effects of S179D-hPRL on all parameters except p53, or activity of the Bax promoter. We conclude that low doses of S179D-hPRL block bFGF-induced ERK signaling and yet activates ERK in a different time frame to elevate p21, and activate the extrinsic pathway. Longer incubations and higher concentrations, however, additionally activate the intrinsic pathway using an alternate intracellular signal. These findings suggest that circulating levels of phosphorylated hPRL may reduce the progression of cancer and, furthermore, that S179D-hPRL may be a useful anti-angiogenic therapeutic. (author)

Tyrosine 842 in the activation loop is required for full transformation by the oncogenic mutant FLT3-ITD.

Science.gov (United States)

Kazi, Julhash U; Chougule, Rohit A; Li, Tianfeng; Su, Xianwei; Moharram, Sausan A; Rupar, Kaja; Marhäll, Alissa; Gazi, Mohiuddin; Sun, Jianmin; Zhao, Hui; Rönnstrand, Lars

2017-07-01

The type III receptor tyrosine kinase FLT3 is frequently mutated in acute myeloid leukemia. Oncogenic FLT3 mutants display constitutive activity leading to aberrant cell proliferation and survival. Phosphorylation on several critical tyrosine residues is known to be essential for FLT3 signaling. Among these tyrosine residues, Y842 is located in the so-called activation loop. The position of this tyrosine residue is well conserved in all receptor tyrosine kinases. It has been reported that phosphorylation of the activation loop tyrosine is critical for catalytic activity for some but not all receptor tyrosine kinases. The role of Y842 residue in FLT3 signaling has not yet been studied. In this report, we show that Y842 is not important for FLT3 activation or ubiquitination but plays a critical role in regulating signaling downstream of the receptor as well as controlling receptor stability. We found that mutation of Y842 in the FLT3-ITD oncogenic mutant background reduced cell viability and increased apoptosis. Furthermore, the introduction of the Y842 mutation in the FLT3-ITD background led to a dramatic reduction in in vitro colony forming capacity. Additionally, mice injected with cells expressing FLT3-ITD/Y842F displayed a significant delay in tumor formation, compared to FLT3-ITD expressing cells. Microarray analysis comparing gene expression regulated by FLT3-ITD versus FLT3-ITD/Y842F demonstrated that mutation of Y842 causes suppression of anti-apoptotic genes. Furthermore, we showed that cells expressing FLT3-ITD/Y842F display impaired activity of the RAS/ERK pathway due to reduced interaction between FLT3 and SHP2 leading to reduced SHP2 activation. Thus, we suggest that Y842 is critical for FLT3-mediated RAS/ERK signaling and cellular transformation.

Protein-Tyrosine Phosphorylation in Bacillus subtilis

DEFF Research Database (Denmark)

Mijakovic, Ivan; Petranovic, Dina; Bottini, N.

2005-01-01

phosphorylation, indicating that this post-translational modifi cation could regulate physiological processes ranging from stress response and exopolysaccharide synthesis to DNA metabolism. Some interesting work in this fi eld was done in Bacillus subtilis , and we here present the current state of knowledge...... on protein-tyrosine phosphorylation in this gram-positive model organism. With its two kinases, two kinase modulators, three phosphatases and at least four different tyrosine-phosphorylated substrates, B. subtilis is the bacterium with the highest number of presently known participants in the global network...

Phosphorylation of Staphylococcus aureus Protein-Tyrosine Kinase Affects the Function of Glucokinase and Biofilm Formation.

Science.gov (United States)

Vasu, Dudipeta; Kumar, Pasupuleti Santhosh; Prasad, Uppu Venkateswara; Swarupa, Vimjam; Yeswanth, Sthanikam; Srikanth, Lokanathan; Sunitha, Manne Mudhu; Choudhary, Abhijith; Sarma, Potukuchi Venkata Gurunadha Krishna

2017-03-01

When Staphylococcus aureus is grown in the presence of high concentration of external glucose, this sugar is phosphorylated by glucokinase (glkA) to form glucose-6-phosphate. This product subsequently enters into anabolic phase, which favors biofilm formation. The presence of ROK (repressor protein, open reading frame, sugar kinase) motif, phosphate-1 and -2 sites, and tyrosine kinase sites in glkA of S. aureus indicates that phosphorylation must regulate the glkA activity. The aim of the present study was to identify the effect of phosphorylation on the function of S. aureus glkA and biofilm formation. Pure glkA and protein-tyrosine kinase (BYK) of S. aureus ATCC 12600 were obtained by fractionating the cytosolic fractions of glkA1 and BYK-1 expressing recombinant clones through nickel metal chelate column. The pure glkA was used as a substrate for BYK and the phosphorylation of glkA was confirmed by treating with reagent A and resolving in SDS-PAGE, as well as staining with reagent A. The kinetic parameters of glkA and phosphorylated glkA were determined spectrophotometrically, and in silico tools were used for validation. S. aureus was grown in brain heart infusion broth, which was supplemented with glucose, and then biofilm units were calculated. Fourfold elevated glkA activity was observed upon the phosphorylation by BYK. Protein-protein docking analysis revealed that glkA structure docked close to the adenosine triphosphate-binding site of BYK structure corroborating the kinetic results. Further, S. aureus grown in the presence of elevated glucose concentration exhibited an increase in the rate of biofilm formation. The elevated function of glkA is an essential requirement for increased biofilm units in S. aureus, a key pathogenic factor that helps its survival and spread the infection.

Carboxamide Spleen Tyrosine Kinase (Syk) Inhibitors: Leveraging Ground State Interactions To Accelerate Optimization

Energy Technology Data Exchange (ETDEWEB)

Ellis, J. Michael; Altman, Michael D.; Cash, Brandon; Haidle, Andrew M.; Kubiak, Rachel L.; Maddess, Matthew L.; Yan, Youwei; Northrup, Alan B. (Merck)

2016-12-08

Optimization of a series of highly potent and kinome selective carbon-linked carboxamide spleen tyrosine kinase (Syk) inhibitors with favorable drug-like properties is described. A pervasive Ames liability in an analogous nitrogen-linked carboxamide series was obviated by replacement with a carbon-linked moiety. Initial efforts lacked on-target potency, likely due to strain induced between the hinge binding amide and solvent front heterocycle. Consideration of ground state and bound state energetics allowed rapid realization of improved solvent front substituents affording subnanomolar Syk potency and high kinome selectivity. These molecules were also devoid of mutagenicity risk as assessed via the Ames test using the TA97a Salmonella strain.

Specific oncogenic activity of the Src-family tyrosine kinase c-Yes in colon carcinoma cells.

Directory of Open Access Journals (Sweden)

Florence Sancier

Full Text Available c-Yes, a member of the Src tyrosine kinase family, is found highly activated in colon carcinoma but its importance relative to c-Src has remained unclear. Here we show that, in HT29 colon carcinoma cells, silencing of c-Yes, but not of c-Src, selectively leads to an increase of cell clustering associated with a localisation of β-catenin at cell membranes and a reduction of expression of β-catenin target genes. c-Yes silencing induced an increase in apoptosis, inhibition of growth in soft-agar and in mouse xenografts, inhibition of cell migration and loss of the capacity to generate liver metastases in mice. Re-introduction of c-Yes, but not c -Src, restores transforming properties of c-Yes depleted cells. Moreover, we found that c-Yes kinase activity is required for its role in β-catenin localisation and growth in soft agar, whereas kinase activity is dispensable for its role in cell migration. We conclude that c-Yes regulates specific oncogenic signalling pathways important for colon cancer progression that is not shared with c-Src.

Spleen tyrosine kinase (Syk), a novel target of curcumin, is required for B lymphoma growth.

Science.gov (United States)

Gururajan, Murali; Dasu, Trivikram; Shahidain, Seif; Jennings, C Darrell; Robertson, Darrell A; Rangnekar, Vivek M; Bondada, Subbarao

2007-01-01

Curcumin (diferuloylmethane), a component of dietary spice turmeric (Curcuma longa), has been shown in recent studies to have therapeutic potential in the treatment of cancer, diabetes, arthritis, and osteoporosis. We investigated the ability of curcumin to modulate the growth of B lymphomas. Curcumin inhibited the growth of both murine and human B lymphoma in vitro and murine B lymphoma in vivo. We also demonstrate that curcumin-mediated growth inhibition of B lymphoma is through inhibition of the survival kinase Akt and its key target Bad. However, in vitro kinase assays show that Akt is not a direct target of curcumin. We identified a novel target for curcumin in B lymphoma viz spleen tyrosine kinase (Syk). Syk is constitutively activated in primary tumors and B lymphoma cell lines and curcumin down-modulates Syk activity accompanied by down-regulation of Akt activation. Moreover, we show that overexpression of Akt, a target of Syk, or Bcl-x(L), a target of Akt can overcome curcumin-induced apoptosis of B lymphoma cells. These observations suggest a novel growth promoting role for Syk in lymphoma cells.

TPX2 in malignantly transformed human bronchial epithelial cells by anti-benzo[a]pyrene-7,8-diol-9,10-epoxide

International Nuclear Information System (INIS)

Zhang Lijuan; Huang He; Deng Luyao; Chu Ming; Xu Lan; Fu Juanling; Zhu Yunlan; Zhang Xiuchun; Liu Shulin; Zhou Zongcan; Wang Yuedan

2008-01-01

In order to elucidate the function of the targeting protein for Xenopus kinesin-like protein 2 (Xklp2) (TPX2) in the malignant transformation of human bronchial epithelial cells induced by anti-benzo[a]pyrene-trans-7, 8-dihydrodiol-9, 10-epoxide (anti-BPDE), TPX2 was characterized in cells at both the gene and the protein levels. TPX2 was present at higher levels in 16HBE-C cells than in 16HBE cells as demonstrated by two-dimensional gel electrophoresis, immunocytochemistry, Western blot analysis and RT-PCR. TPX2 was also detected in lung squamous-cell carcinoma tissues by immunohistochemistry, but not in normal lung tissues. Depression of TPX2 by RNA interference in 16HBE-C cells led to a decrease in cell proliferation, S-phase cell cycle arrest and cell apoptosis. Abnormal TPX2 tyrosine phosphorylation was detected in 16HBE-C cells, and this could be inhibited, to different degrees, by tyrosine kinase inhibitors. Inhibiting tyrosine phosphorylation in 16HBE-C cells by three selected tyrosine protein kinase inhibitors, tyrphostin 47, AG112 and AG555, caused G 0 /G 1 -phase cell cycle arrest. Our results suggest that anti-BPDE can cause the over-expression of TPX2 and its aberrant tyrosine phosphorylation. Misregulation of TPX2 affects the cell cycle state, proliferation rates and apoptosis

Neratinib, A Novel HER2-Targeted Tyrosine Kinase Inhibitor.

Science.gov (United States)

Tiwari, Shruti Rakesh; Mishra, Prasun; Abraham, Jame

2016-10-01

HER2 gene amplification and receptor overexpression is identified in 20% to 25% of human breast cancers. Use of targeted therapy for HER2-amplified breast cancer has led to improvements in disease-free and overall survival in this subset of patients. Neratinib is an oral pan HER inhibitor, that irreversibly inhibits the tyrosine kinase activity of epidermal growth factor receptor (EGFR or HER1), HER2, and HER4, which leads to reduced phosphorylation and activation of downstream signaling pathways. Neratinib is currently being tested in a number of clinical trials for its safety and efficacy in lung cancer, and colorectal, bladder, and breast cancers. In this review we discuss the available phase I, II, and III data for use of neratinib in the metastatic, adjuvant, neoadjuvant, and extended adjuvant settings along with the ongoing clinical trials of neratinib in breast cancer. We also elaborate on the side effect profile of this relatively new drug and provide guidelines for its use in clinical practice. Copyright © 2016 Elsevier Inc. All rights reserved.

Protein tyrosine phosphatase receptor delta acts as a neuroblastoma tumor suppressor by destabilizing the aurora kinase a oncogene

LENUS (Irish Health Repository)

Meehan, Maria

2012-02-05

Abstract Background Protein tyrosine phosphatase receptor delta (PTPRD) is a member of a large family of protein tyrosine phosphatases which negatively regulate tyrosine phosphorylation. Neuroblastoma is a major childhood cancer arising from precursor cells of the sympathetic nervous system which is known to acquire deletions and alterations in the expression patterns of PTPRD, indicating a potential tumor suppressor function for this gene. The molecular mechanism, however, by which PTPRD renders a tumor suppressor effect in neuroblastoma is unknown. Results As a molecular mechanism, we demonstrate that PTPRD interacts with aurora kinase A (AURKA), an oncogenic protein that is over-expressed in multiple forms of cancer, including neuroblastoma. Ectopic up-regulation of PTPRD in neuroblastoma dephosphorylates tyrosine residues in AURKA resulting in a destabilization of this protein culminating in interfering with one of AURKA\\'s primary functions in neuroblastoma, the stabilization of MYCN protein, the gene of which is amplified in approximately 25 to 30% of high risk neuroblastoma. Conclusions PTPRD has a tumor suppressor function in neuroblastoma through AURKA dephosphorylation and destabilization and a downstream destabilization of MYCN protein, representing a novel mechanism for the function of PTPRD in neuroblastoma.

Phosphorylation of actin-binding protein (ABP-280; filamin) by tyrosine kinase p56lck modulates actin filament cross-linking.

Science.gov (United States)

Pal Sharma, C; Goldmann, Wolfgang H

2004-01-01

Actin-binding protein (ABP-280; filamin) is a phosphoprotein present in the periphery of the cytoplasm where it can cross-link actin filaments, associate with lipid membranes, and bind to membrane surface receptors. Given its function and localization in the cell, we decided to investigate the possibility of whether it serves as substrate for p56lck, a lymphocyte-specific member of the src family of protein tyrosine kinases associated with cell surface glycoproteins. The interaction of p56lck with membrane glycoproteins is important for cell development and functional activation. Here, we show that purified p56lck interacts and catalyzes in vitro kinase reactions. Tyrosine phosphorylation by p56lck is restricted to a single peptide of labeled ABP-280 shown by protease digest. The addition of phorbol ester to cells results in the inhibition of phosphorylation of ABP-280 by p56lck. These results show a decrease in phosphorylation suggesting conformationally induced regulation. Dynamic light scattering confirmed increased actin filament cross-linking due to phosphorylation of ABP-280 by p56lck.

Crosstalk between G protein-coupled receptors (GPCRs and tyrosine kinase receptor (TXR in the heart after morphine withdrawal

Directory of Open Access Journals (Sweden)

Pilar eAlmela

2013-12-01

Full Text Available G protein-coupled receptors (GPCRs comprise a large family of membrane receptors involved in signal transduction. These receptors are linked to a variety of physiological and biological processes such as regulation of neurotransmission, growth and cell differentiation among others. Some of the effects of GPCRs are known to be mediated by the activation of mitogen-activated extracellular kinase (MAPK pathways. Cross-talk among various signal pathways plays an important role in activation of intracellular and intranuclear signal transduction cascades. Naloxone-induced morphine withdrawal leads to an up-regulation of adenyl cyclase-mediated signalling, resulting in high expression of protein kinase (PK A. In addition, there is also an increased expression of extracellular signal regulated kinase (ERK, one member of MAPK. For this reason, the crosstalk between these GPCRs and receptors with tyrosine kinase activity (TKR can be considered a possible mechanism for adaptive changes that occurs after morphine withdrawal. Morphine withdrawal activates ERK1/2 and phosphorylated tyrosine hydroxylase (TH at Ser31 in the right and left ventricle. When N-(2-guanidinoethyl-5-isoquinolinesulfonamide (HA-1004, a PKA inhibitor was infused, the ability of morphine withdrawal to activate ERK, which phosphorylates TH at Ser31, was reduced. The present finding demonstrated that the enhancement of ERK1/2 expression and the phosphorylation state of TH at Ser31 during morphine withdrawal are dependent on PKA and suggest cross-talk between PKA and ERK1/2 transduction pathway mediating morphine withdrawal-induced activation of TH. Increasing understanding of the mechanisms that interconnect the two pathway regulated by GPCRs and TKRs may facilitate the design of new therapeutic strategies.

Emerging paradigms and questions on pro-angiogenic bone marrow-derived myelomonocytic cells.

Science.gov (United States)

Laurent, Julien; Touvrey, Cédric; Botta, Francesca; Kuonen, François; Ruegg, Curzio

2011-01-01

Cancer-related inflammation has emerged in recent years as a major event contributing to tumor angiogenesis, tumor progression and metastasis formation. Bone marrow-derived and inflammatory cells promote tumor angiogenesis by providing endothelial progenitor cells that differentiate into mature endothelial cells, and by secreting pro-angiogenic factors and remodeling the extracellular matrix to stimulate angiogenesis though paracrine mechanisms. Several bone marrow-derived myelonomocytic cells, including monocytes and macrophages, have been identified and characterized by several laboratories in recent years. While the central role of these cells in promoting tumor angiogenesis, tumor progression and metastasis is nowadays well established, many questions remain open and new ones are emerging. These include the relationship between their phenotype and function, the mechanisms of pro-angiogenic programming, their contribution to resistance to anti-angiogenic treatments and to metastasis and their potential clinical use as biomarkers of angiogenesis and anti-angiogenic therapies. Here, we will review phenotypical and functional aspects of bone marrow-derived myelonomocytic cells and discuss some of the current outstanding questions.

Sequential treatment of tyrosine kinase inhibitors and chemotherapy for EGFR-mutated non-small cell lung cancer: a meta-analysis of Phase III trials

Directory of Open Access Journals (Sweden)

Zhang Y

2013-11-01

Full Text Available Yiliang Zhang,1,* Yihua Sun,1,* Lei Wang,1 Ting Ye,1 Yunjian Pan,1 Haichuan Hu,1 Yongfu Yu,2 Naiqing Zhao,2 Yanyan Song,3 David Garfield,4 Haiquan Chen1 1Department of Thoracic Surgery, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, 2Department of Biostatistics, School of Public Health, Fudan University, 3Department of Pharmacology and Biostatistics, Institute of Medical Science, Shanghai Jiaotong University School of Medicine, 4ProMed Cancer Centers, Shanghai, People’s Republic of China *These authors contributed equally to this work Background: This aim of this study was to compare the efficacy of first-line tyrosine kinase inhibitor therapy followed, upon progression, by chemotherapy with the reverse sequence in patients with EGFR-mutated non-small cell lung cancer (NSCLC in terms of overall survival. Methods: We performed a meta-analysis of studies that met the following criteria: Phase III clinical trial comparing the sequencing of epidermal growth factor receptor (EGFR tyrosine kinase inhibitors with chemotherapy in the treatment of advanced EGFR-mutated NSCLC; activating mutations reported; and availability of hazard ratio estimates with 95% confidence intervals (CIs for overall survival. Results: Six clinical trials were included in this study. The pooled hazard ratio for overall survival of the EGFR-mutated population that completed sequential treatment was 1.03 (95% CI 0.86–1.22, P=0.776. There was no statistically significant heterogeneity between the studies (tau2 =0; I2=0, 95% CI 0–0.37, P=0.548. Evidence of marked publication bias for the two treatment sequences was insufficient (P=0.145. Conclusion: In patients with advanced NSCLC and activating EGFR mutations, first-line chemotherapy followed upon progression by a tyrosine kinase inhibitor was not inferior in terms of overall survival compared with the inverse sequence. This may serve as an indication that

Ginsenoside-Rg{sub 1} induces angiogenesis by the inverse regulation of MET tyrosine kinase receptor expression through miR-23a

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Kwok, Hoi-Hin [Dr. Gilbert Hung Ginseng Laboratory, Faculty of Science, Hong Kong Baptist University, Hong Kong SAR (China); Chan, Lai-Sheung [Department of Biology, Faculty of Science, Hong Kong Baptist University, Hong Kong SAR (China); Poon, Po-Ying [Dr. Gilbert Hung Ginseng Laboratory, Faculty of Science, Hong Kong Baptist University, Hong Kong SAR (China); Yue, Patrick Ying-Kit [Dr. Gilbert Hung Ginseng Laboratory, Faculty of Science, Hong Kong Baptist University, Hong Kong SAR (China); Department of Biology, Faculty of Science, Hong Kong Baptist University, Hong Kong SAR (China); Wong, Ricky Ngok-Shun, E-mail: rnswong@hkbu.edu.hk [Dr. Gilbert Hung Ginseng Laboratory, Faculty of Science, Hong Kong Baptist University, Hong Kong SAR (China); Department of Biology, Faculty of Science, Hong Kong Baptist University, Hong Kong SAR (China)

2015-09-15

Therapeutic angiogenesis has been implicated in ischemic diseases and wound healing. Ginsenoside-Rg{sub 1} (Rg{sub 1}), one of the most abundant active components of ginseng, has been demonstrated as an angiogenesis-stimulating compound in different models. There is increasing evidence implicating microRNAs (miRNAs), a group of non-coding RNAs, as important regulators of angiogenesis, but the role of microRNAs in Rg{sub 1}-induced angiogenesis has not been fully explored. In this report, we found that stimulating endothelial cells with Rg{sub 1} could reduce miR-23a expression. In silico experiments predicted hepatocyte growth factor receptor (MET), a well-established mediator of angiogenesis, as the target of miR-23a. Transfection of the miR-23a precursor or inhibitor oligonucleotides validated the inverse relationship of miR-23a and MET expression. Luciferase reporter assays further confirmed the interaction between miR-23a and the MET mRNA 3′-UTR. Intriguingly, ginsenoside-Rg{sub 1} was found to increase MET protein expression in a time-dependent manner. We further demonstrated that ginsenoside-Rg{sub 1}-induced angiogenic activities were indeed mediated through the down-regulation of miR-23a and subsequent up-regulation of MET protein expression, as confirmed by gain- and loss-of-function angiogenic experiments. In summary, our results demonstrated that ginsenoside-Rg{sub 1} could induce angiogenesis by the inverse regulation of MET tyrosine kinase receptor expression through miR-23a. This study has broadened our understanding of the non-genomic effects of ginsenoside-Rg{sub 1,} and provided molecular evidence that warrant further development of natural compound as novel angiogenesis-promoting therapy. - Highlights: • Therapeutic angiogenesis has been implicated in ischemic diseases and wound healing. • Ginsenoside-Rg{sub 1} (Rg{sub 1}) has been demonstrated as an angiogenesis-stimulating compound. • We found that Rg{sub 1} induces angiogenesis by

Combined treatment of tyrosine kinase inhibitor labeled gold nanorod encapsulated albumin with laser thermal ablation in a renal cell carcinoma model

Science.gov (United States)

This manuscript served to characterize and evaluate Human Serum Albumin-encapsulated Nanoparticles (NPs) for drug delivery of a tyrosine kinase inhibitor combined with induction of photothermal ablation (PTA) combination therapy of Renal Cell Carcinoma (RCC). RCC is the most common type of kidney c...

Dialkoxyquinazolines: Screening Epidermal Growth Factor ReceptorTyrosine Kinase Inhibitors for Potential Tumor Imaging Probes

Energy Technology Data Exchange (ETDEWEB)

VanBrocklin, Henry F.; Lim, John K.; Coffing, Stephanie L.; Hom,Darren L.; Negash, Kitaw; Ono, Michele Y.; Hanrahan, Stephen M.; Taylor,Scott E.; Vanderpoel, Jennifer L.; Slavik, Sarah M.; Morris, Andrew B.; Riese II, David J.

2005-09-01

The epidermal growth factor receptor (EGFR), a long-standingdrug development target, is also a desirable target for imaging. Sixteendialkoxyquinazoline analogs, suitable for labeling with positron-emittingisotopes, have been synthesized and evaluated in a battery of in vitroassays to ascertain their chemical and biological properties. Thesecharacteristics provided the basis for the adoption of a selection schemato identify lead molecules for labeling and in vivo evaluation. A newEGFR tyrosine kinase radiometric binding assay revealed that all of thecompounds possessed suitable affinity (IC50 = 0.4 - 51 nM) for the EGFRtyrosine kinase. All of the analogs inhibited ligand-induced EGFRtyrosine phosphorylation (IC50 = 0.8 - 20 nM). The HPLC-estimatedoctanol/water partition coefficients ranged from 2.0-5.5. Four compounds,4-(2'-fluoroanilino)- and 4-(3'-fluoroanilino)-6,7-diethoxyquinazoline aswell as 4-(3'-chloroanilino)- and4-(3'-bromoanilino)-6,7-dimethoxyquinazoline, possess the bestcombination of characteristics that warrant radioisotope labeling andfurther evaluation in tumor-bearing mice.

The Use of EGFR Tyrosine Kinase Inhibitors in EGFR Wild-Type Non-Small-Cell Lung Cancer.

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Stinchcombe, Thomas E

2016-04-01

The objective response rate and progression-free survival observed with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) in patients with metastatic epidermal growth factor receptor (EGFR) wild-type non-small cell lung cancer (NSCLC) are modest. The adverse events associated with EGFR TKIs are manageable but they must be considered in the context of the limited efficacy. The development of anti-PD-1 immunotherapy as second-line therapy has reduced the role of EGFR TKIs in EGFR wild-type NSCLC. Recently, there has been increased recognition of the benefit of the earlier integration of palliative care and symptom management, and this is reasonable alternative to treatment with an EGFR TKI for many patients. My practice pattern for patients with EGFR wild-type NSCLC is platinum-based chemotherapy as first-line therapy, immunotherapy as second-line therapy, and single-agent chemotherapy as third-line therapy for patients with preserved performance status who want to pursue further therapy. Only a small proportion of patients are eligible for fourth-line therapy, and I prefer to enroll them in clinical trials rather than use EGFR TKIs. I suspect that the use of EGFR TKIs in clinical use and as a comparator arm for clinical trials will continue to decline over the next several years.

Key endothelial cell angiogenic mechanisms are stimulated by the circulating milieu in sickle cell disease and attenuated by hydroxyurea

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Lopes, Flavia C. M.; Traina, Fabiola; Almeida, Camila B.; Leonardo, Flavia C.; Franco-Penteado, Carla F.; Garrido, Vanessa T.; Colella, Marina P.; Soares, Raquel; Olalla-Saad, Sara T.; Costa, Fernando F.; Conran, Nicola

2015-01-01

As hypoxia-induced inflammatory angiogenesis may contribute to the manifestations of sickle cell disease, we compared the angiogenic molecular profiles of plasma from sickle cell disease individuals and correlated these with in vitro endothelial cell-mediated angiogenesis-stimulating activity and in vivo neovascularization. Bioplex demonstrated that plasma from patients with steady-state sickle cell anemia contained elevated concentrations of pro-angiogenic factors (angiopoietin-1, basic fibroblast growth factor, vascular endothelial growth factor, vascular endothelial growth factor-D and placental growth factor) and displayed potent pro-angiogenic activity, significantly increasing endothelial cell proliferation, migration and capillary-like structure formation. In vivo neovascularization of Matrigel plugs was significantly greater in sickle cell disease mice than in non-sickle cell disease mice, consistent with an up-regulation of angiogenesis in the disease. In plasma from patients with hemoglobin SC disease without proliferative retinopathy, anti-angiogenic endostatin and thrombospondin-2 were significantly elevated. In contrast, plasma from hemoglobin SC individuals with proliferative retinopathy had a pro-angiogenic profile and more significant effects on endothelial cell proliferation and capillary formation than plasma from patients without retinopathy. Hydroxyurea therapy was associated with significant reductions in plasma angiogenic factors and inhibition of endothelial cell-mediated angiogenic mechanisms and neovascularization. Thus, individuals with sickle cell anemia or hemoglobin SC disease with retinopathy present a highly angiogenic circulating milieu, capable of stimulating key endothelial cell-mediated angiogenic mechanisms. Combination anti-angiogenic therapy to prevent the progression of unregulated neovascularization and associated manifestations in sickle cell disease, such as pulmonary hypertension, may be indicated; furthermore, the

Aminopeptidase N inhibition could be involved in the anti-angiogenic effect of dobesilates

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Farsa Oldřich

2015-01-01

Full Text Available Calcium, magnesium and zinc 2,5-dihydroxybenzenesulfonates (dobesilates were synthesized by sulfonation of hydroquinone with sulfuric acid under mild conditions. To form the salts, neutralization with calcium carbonate followed by cation exchange by means of magnesium or zinc sulfates was performed. The dobesilates were characterized by standard spectral methods and by AAS for metal content and then tested for inhibitory activity against aminopeptidase N. Calcium and magnesium 2,5-dihydroxybenzene sulfonates exhibited rather weak inhibitory activity to aminopeptidase N as demonstrated by IC50 values of 978.0 and 832.1 mmol l-1 respectively while zinc 2,5-dihydroxybenzene sulfonate reached the more significant inhibitory activity characterized by IC50 77.4 mmol l-1. The inhibitory activity results suggest that the inhibition of aminopeptidase N could play a role in the anti-angiogenic activity of 2,5-dihydroxybenzenesulfonates.

Mel-18, a mammalian Polycomb gene, regulates angiogenic gene expression of endothelial cells.

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Jung, Ji-Hye; Choi, Hyun-Jung; Maeng, Yong-Sun; Choi, Jung-Yeon; Kim, Minhyung; Kwon, Ja-Young; Park, Yong-Won; Kim, Young-Myeong; Hwang, Daehee; Kwon, Young-Guen

2010-10-01

Mel-18 is a mammalian homolog of Polycomb group (PcG) genes. Microarray analysis revealed that Mel-18 expression was induced during endothelial progenitor cell (EPC) differentiation and correlates with the expression of EC-specific protein markers. Overexpression of Mel-18 promoted EPC differentiation and angiogenic activity of ECs. Accordingly, silencing Mel-18 inhibited EC migration and tube formation in vitro. Gene expression profiling showed that Mel-18 regulates angiogenic genes including kinase insert domain receptor (KDR), claudin 5, and angiopoietin-like 2. Our findings demonstrate, for the first time, that Mel-18 plays a significant role in the angiogenic function of ECs by regulating endothelial gene expression. Copyright © 2010 Elsevier Inc. All rights reserved.

Relation between circulating angiotensin II type 1 receptor agonistic autoantibodies and soluble fms-like tyrosine kinase 1 in the pathogenesis of preeclampsia

NARCIS (Netherlands)

H. Stepan (Holger); R. Faber (Renaldo); N. Wessel; G. Wallukat (Gerd); H.P. Schultheiss (Heinz-Peter); T. Walther (Thomas)

2006-01-01

textabstractContext: Placental and circulatory soluble fms-like tyrosine kinase 1 (sFlt1) has proven to be elevated in pregnant women with preeclampsia, a disease characterized by hypertension, proteinuria, and endothelial dysfunction. Recent studies also demonstrated an autoantibody against the

Molecular Mechanisms of SH2- and PTB-Domain-Containing Proteins in Receptor Tyrosine Kinase Signaling

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Wagner, Melany J.; Stacey, Melissa M.; Liu, Bernard A.; Pawson, Tony

2013-01-01

Intracellular signaling is mediated by reversible posttranslational modifications (PTMs) that include phosphorylation, ubiquitination, and acetylation, among others. In response to extracellular stimuli such as growth factors, receptor tyrosine kinases (RTKs) typically dimerize and initiate signaling through phosphorylation of their cytoplasmic tails and downstream scaffolds. Signaling effectors are recruited to these phosphotyrosine (pTyr) sites primarily through Src homology 2 (SH2) domains and pTyr-binding (PTB) domains. This review describes how these conserved domains specifically recognize pTyr residues and play a major role in mediating precise downstream signaling events. PMID:24296166

Molecular mechanisms of SH2- and PTB-domain-containing proteins in receptor tyrosine kinase signaling.

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Wagner, Melany J; Stacey, Melissa M; Liu, Bernard A; Pawson, Tony

2013-12-01

Intracellular signaling is mediated by reversible posttranslational modifications (PTMs) that include phosphorylation, ubiquitination, and acetylation, among others. In response to extracellular stimuli such as growth factors, receptor tyrosine kinases (RTKs) typically dimerize and initiate signaling through phosphorylation of their cytoplasmic tails and downstream scaffolds. Signaling effectors are recruited to these phosphotyrosine (pTyr) sites primarily through Src homology 2 (SH2) domains and pTyr-binding (PTB) domains. This review describes how these conserved domains specifically recognize pTyr residues and play a major role in mediating precise downstream signaling events.

Antitumor activity of TY-011 against gastric cancer by inhibiting Aurora A, Aurora B and VEGFR2 kinases

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Wang Liu

2016-11-01

Full Text Available Abstract Background Overexpression of Aurora A and B has been reported in a wide range of tumor types, including gastric cancer. Anti-angiogenesis has been considered as an important therapeutic modality in advanced gastric cancer. Here we identified a novel compound TY-011 with promising antitumor activity by targeting mitotic kinases (Aurora A and B and angiogenic receptor tyrosine kinase (VEGFR2. Methods HTRF® KinEASE™ assay was used to detect the effect of TY-011 against Aurora A, Aurora B and VEGFR2 activities. Docking simulation study was performed to predict the binding mode of TY-011 with Aurora A and B kinases. CCK-8 assay was used to test cell growth. Cell cycle and cell apoptosis was analyzed by flow cytometry. Gastric cancer cell xenograft mouse models were used for in vivo study. TUNEL kit was used to determine the apoptosis of tumor tissues. Immunohistochemistry analysis and HUVEC tube formation assay were performed to determine the anti-angiogenesis ability. Immunofluorescence and western blot were used to test protein expression. Results TY-011 was identified as a potential Aurora A and B inhibitor by HTRF® KinEASE™ assay. It effectively inhibited cellular Aurora A and B activities in a concentration-dependent manner. TY-011 occupied the ATP-binding site of both Aurora A and B kinases. TY-011 demonstrated prominent inhibitory effects on proliferation of gastric cancer cells. TY-011 treatment induced an obvious accumulation of cells at G2/M phase and a modest increase of cells with >4 N DNA content, which then underwent apoptosis. Meaningfully, orally administration of TY-011 demonstrated superior efficacy against the tumor growth in gastric cancer cell xenograft, with ~90% inhibition rate and 100% tumor regression at 9 mg/kg dose, and TY-011 did not affect the body weight of mice. Interestingly, we observed that TY-011 also antagonized tumor angiogenesis by targeting VEGFR2 kinase. Conclusions These results indicate that

Genistein and tyrphostin AG556 decrease ultra-rapidly activating delayed rectifier K+ current of human atria by inhibiting EGF receptor tyrosine kinase.

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Xiao, Guo-Sheng; Zhang, Yan-Hui; Wu, Wei; Sun, Hai-Ying; Wang, Yan; Li, Gui-Rong

2017-03-01

The ultra-rapidly activating delayed rectifier K + current I Kur (encoded by K v 1.5 or KCNA5) plays an important role in human atrial repolarization. The present study investigates the regulation of this current by protein tyrosine kinases (PTKs). Whole-cell patch voltage clamp technique and immunoprecipitation and Western blotting analysis were used to investigate whether the PTK inhibitors genistein, tyrphostin AG556 (AG556) and PP2 regulate human atrial I Kur and hKv1.5 channels stably expressed in HEK 293 cells. Human atrial I Kur was decreased by genistein (a broad-spectrum PTK inhibitor) and AG556 (a highly selective EGFR TK inhibitor) in a concentration-dependent manner. Inhibition of I Kur induced by 30 μM genistein or 10 μM AG556 was significantly reversed by 1 mM orthovanadate (a protein tyrosine phosphatase inhibitor). Similar results were observed in HEK 293 cells stably expressing hK v 1.5 channels. On the other hand, the Src family kinase inhibitor PP2 (1 μM) slightly enhanced I Kur and hK v 1.5 current, and the current increase was also reversed by orthovanadate. Immunoprecipitation and Western blotting analysis showed that genistein, AG556, and PP2 decreased tyrosine phosphorylation of hK v 1.5 channels and that the decrease was countered by orthovanadate. The PTK inhibitors genistein and AG556 decrease human atrial I Kur and cloned hK v 1.5 channels by inhibiting EGFR TK, whereas the Src kinase inhibitor PP2 increases I Kur and hK v 1.5 current. These results imply that EGFR TK and the soluble Src kinases may have opposite effects on human atrial I Kur . © 2017 The British Pharmacological Society.

Syk Tyrosine Kinase Acts as a Pancreatic Adenocarcinoma Tumor Suppressor by Regulating Cellular Growth and Invasion

OpenAIRE

Layton, Tracy; Stalens, Cristel; Gunderson, Felizza; Goodison, Steve; Silletti, Steve

2009-01-01

We have identified the nonreceptor tyrosine kinase syk as a marker of differentiation/tumor suppressor in pancreatic ductal adenocarcinoma (PDAC). Syk expression is lost in poorly differentiated PDAC cells in vitro and in situ, and stable reexpression of syk in endogenously syk-negative Panc1 (Panc1/syk) cells retarded their growth in vitro and in vivo and reduced anchorage-independent growth in vitro. Panc1/syk cells exhibited a more differentiated morphology and down-regulated cyclin D1, ak...

CS2164, a novel multi-target inhibitor against tumor angiogenesis, mitosis and chronic inflammation with anti-tumor potency.

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Zhou, You; Shan, Song; Li, Zhi-Bin; Xin, Li-Jun; Pan, De-Si; Yang, Qian-Jiao; Liu, Ying-Ping; Yue, Xu-Peng; Liu, Xiao-Rong; Gao, Ji-Zhou; Zhang, Jin-Wen; Ning, Zhi-Qiang; Lu, Xian-Ping

2017-03-01

Although inhibitors targeting tumor angiogenic pathway have provided improvement for clinical treatment in patients with various solid tumors, the still very limited anti-cancer efficacy and acquired drug resistance demand new agents that may offer better clinical benefits. In the effort to find a small molecule potentially targeting several key pathways for tumor development, we designed, discovered and evaluated a novel multi-kinase inhibitor, CS2164. CS2164 inhibited the angiogenesis-related kinases (VEGFR2, VEGFR1, VEGFR3, PDGFRα and c-Kit), mitosis-related kinase Aurora B and chronic inflammation-related kinase CSF-1R in a high potency manner with the IC 50 at a single-digit nanomolar range. Consequently, CS2164 displayed anti-angiogenic activities through suppression of VEGFR/PDGFR phosphorylation, inhibition of ligand-dependent cell proliferation and capillary tube formation, and prevention of vasculature formation in tumor tissues. CS2164 also showed induction of G2/M cell cycle arrest and suppression of cell proliferation in tumor tissues through the inhibition of Aurora B-mediated H3 phosphorylation. Furthermore, CS2164 demonstrated the inhibitory effect on CSF-1R phosphorylation that led to the suppression of ligand-stimulated monocyte-to-macrophage differentiation and reduced CSF-1R + cells in tumor tissues. The in vivo animal efficacy studies revealed that CS2164 induced remarkable regression or complete inhibition of tumor growth at well-tolerated oral doses in several human tumor xenograft models. Collectively, these results indicate that CS2164 is a highly selective multi-kinase inhibitor with potent anti-tumor activities against tumor angiogenesis, mitosis and chronic inflammation, which may provide the rationale for further clinical assessment of CS2164 as a therapeutic agent in the treatment of cancer. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Friends Turned Foes: Angiogenic Growth Factors beyond Angiogenesis.

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Matkar, Pratiek N; Ariyagunarajah, Ramya; Leong-Poi, Howard; Singh, Krishna K

2017-10-02

Angiogenesis, the formation of new blood vessels from pre-existing ones is a biological process that ensures an adequate blood flow is maintained to provide the cells with a sufficient supply of nutrients and oxygen within the body. Numerous soluble growth factors and inhibitors, cytokines, proteases as well as extracellular matrix proteins and adhesion molecules stringently regulate the multi-factorial process of angiogenesis. The properties and interactions of key angiogenic molecules such as vascular endothelial growth factors (VEGFs), fibroblast growth factors (FGFs) and angiopoietins have been investigated in great detail with respect to their molecular impact on angiogenesis. Since the discovery of angiogenic growth factors, much research has been focused on their biological actions and their potential use as therapeutic targets for angiogenic or anti-angiogenic strategies in a context-dependent manner depending on the pathologies. It is generally accepted that these factors play an indispensable role in angiogenesis. However, it is becoming increasingly evident that this is not their only role and it is likely that the angiogenic factors have important functions in a wider range of biological and pathological processes. The additional roles played by these molecules in numerous pathologies and biological processes beyond angiogenesis are discussed in this review.

Autophosphorylation of JAK2 on tyrosines 221 and 570 regulates its activity.

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Argetsinger, Lawrence S; Kouadio, Jean-Louis K; Steen, Hanno; Stensballe, Allan; Jensen, Ole N; Carter-Su, Christin

2004-06-01

The tyrosine kinase JAK2 is a key signaling protein for at least 20 receptors in the cytokine/hematopoietin receptor superfamily and is a component of signaling by insulin receptor and several G-protein-coupled receptors. However, there is only limited knowledge of the physical structure of JAK2 or which of the 49 tyrosines in JAK2 are autophosphorylated. In this study, mass spectrometry and two-dimensional peptide mapping were used to determine that tyrosines 221, 570, and 1007 in JAK2 are autophosphorylated. Phosphorylation of tyrosine 570 is particularly robust. In response to growth hormone, JAK2 was rapidly and transiently phosphorylated at tyrosines 221 and 570, returning to basal levels by 60 min. Analysis of the sequences surrounding tyrosines 221 and 570 in JAK2 and tyrosines in other proteins that are phosphorylated in response to ligands that activate JAK2 suggests that the YXX[L/I/V] motif is one of the motifs recognized by JAK2. Experiments using JAK2 with tyrosines 221 and 570 mutated to phenylalanine suggest that tyrosines 221 and 570 in JAK2 may serve as regulatory sites in JAK2, with phosphorylation of tyrosine 221 increasing kinase activity and phosphorylation of tyrosine 570 decreasing kinase activity and thereby contributing to rapid termination of ligand activation of JAK2.

Novel autophosphorylation sites of Src family kinases regulate kinase activity and SH2 domain-binding capacity.

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Weir, Marion E; Mann, Jacqueline E; Corwin, Thomas; Fulton, Zachary W; Hao, Jennifer M; Maniscalco, Jeanine F; Kenney, Marie C; Roman Roque, Kristal M; Chapdelaine, Elizabeth F; Stelzl, Ulrich; Deming, Paula B; Ballif, Bryan A; Hinkle, Karen L

2016-04-01

Src family tyrosine kinases (SFKs) are critical players in normal and aberrant biological processes. While phosphorylation importantly regulates SFKs at two known tyrosines, large-scale phosphoproteomics have revealed four additional tyrosines commonly phosphorylated in SFKs. We found these novel tyrosines to be autophosphorylation sites. Mimicking phosphorylation at the C-terminal site to the activation loop decreased Fyn activity. Phosphomimetics and direct phosphorylation at the three SH2 domain sites increased Fyn activity while reducing phosphotyrosine-dependent interactions. While 68% of human SH2 domains exhibit conservation of at least one of these tyrosines, few have been found phosphorylated except when found in cis to a kinase domain. © 2016 Federation of European Biochemical Societies.

Os inibidores de tirosino quinase de segunda geração The inhibitors of tyrosine kinase

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Márcia T. Delamain

2008-04-01

Full Text Available O imatinibe tem sido confirmado como terapia de primeira linha para a Leucemia Mielóide Crônica (LMC por apresentar respostas duradouras na maior parte dos pacientes, principalmente nos que se encontram em fase precoce da doença. Entretanto, resistência ou intolerância ao imatinibe podem ocorrer. A resistência ao imatinibe ocorre com muito mais freqüência em fases mais avançadas da doença, sendo a causa mais comum o desenvolvimento de mutações no sítio BCR-ABL. Em face deste problema, novos inibidores de tirosino quinase têm sido desenvolvidos, com maior potência, diminuindo assim a chance de desenvolvimento de resistência ao tratamento. O nilotinibe e o dasatinibe são dois exemplos de inibidores de segunda geração de tirosino quinase recentemente aprovados. Ambos têm demonstrado excelentes resultados em pacientes que desenvolvem resistência ou são intolerantes ao imatinibe.Despite the success with imatinib as the first choice treatment of chronic myeloid leukemia (CML, there is still a subset of patients that do not respond optimally to or are intolerant of this drug or lose response. Imatinib resistance can occur at any phase, but it is more frequent in advanced phases, with mutations in the BCR-ABL kinase domain being the most common mechanism of resistance. More potent tyrosine kinase inhibitors have been developed that can overcome resistance to imatinib. Nilotinib and dasatinib are good examples of new tyrosine kinase inhibitors that are available. With these new agents, patients who develop imatinib resistance or those unable to tolerate imatinib treatment can achieve significant clinical responses.

Bruton's tyrosine kinase is essential for hydrogen peroxide-induced calcium signaling.

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Qin, S; Chock, P B

2001-07-10

Using Btk-deficient DT40 cells and the transfectants expressing wild-type Btk or Btk mutants in either kinase (Arg(525) to Gln), Src homology 2 (SH2, Arg(307) to Ala), or pleckstrin homology (PH, Arg(28) to Cys) domains, we investigated the roles and structure-function relationships of Btk in hydrogen peroxide-induced calcium mobilization. Our genetic evidence showed that Btk deficiency resulted in a significant reduction in hydrogen peroxide-induced calcium response. This impaired calcium signaling is correlated with the complete elimination of IP3 production and the significantly reduced tyrosine phosphorylation of PLCgamma2 in Btk-deficient DT40 cells. All of these defects were fully restored by the expression of wild-type Btk in Btk-deficient DT40 cells. The data from the point mutation study revealed that a defect at any one of the three functional domains would prevent a full recovery of Btk-mediated hydrogen peroxide-induced intracellular calcium mobilization. However, mutation at either the SH2 or PH domain did not affect the hydrogen peroxide-induced activation of Btk. Mutation at the SH2 domain abrogates both IP3 generation and calcium release, while the mutant with the nonfunctional PH domain can partially activate PLCgamma2 and catalyze IP3 production but fails to produce significant calcium mobilization. Thus, these observations suggest that Btk-dependent tyrosine phosphorylation of PLCgamma2 is required but not sufficient for hydrogen peroxide-induced calcium mobilization. Furthermore, hydrogen peroxide stimulates a Syk-, but not Btk-, dependent tyrosine phosphorylation of B cell linker protein BLNK. The overall results, together with those reported earlier [Qin et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 7118], are consistent with the notion that functional SH2 and PH domains are required for Btk to form a complex with PLCgamma2 through BLNK in order to position the Btk, PLCgamma2, and phosphatidylinositol 4,5-bisphosphate in close proximity for

Patients harboring EGFR mutation after primary resistance to crizotinib and response to EGFR-tyrosine kinase inhibitor

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Wang WX

2016-01-01

Full Text Available Wenxian Wang,1 Xiaowen Jiang,1 Zhengbo Song,1,2 Yiping Zhang1,2 1Department of Chemotherapy, Zhejiang Cancer Hospital, 2Key Laboratory Diagnosis and Treatment Technology on Thoracic Oncology, Hangzhou, Zhejiang, People’s Republic of China Abstract: Anaplastic lymphoma kinase (ALK rearrangement lung cancer responds to ALK tyrosine kinase inhibitors. It is known that many cases ultimately acquired resistance to crizotinib. However, a case of primary resistance is rare. We present a case of harboring exon 19 deletion in epidermal growth factor receptor in ALK rearranged lung adenocarcinoma, who experienced a partial tumor response to icotinib after failure with crizotinib therapy and chemotherapy. Considering the partial response, we conclude that it is important to find the cause of resistance to crizotinib. We detected gene mutations with plasma by the next-generation sequencing; the next-generation sequencing demonstrates an attractive system to identify mutations improving the outcome of patients with a deadly disease. Keywords: non-small cell lung cancer, anaplastic lymphoma kinase, crizotinib, epidermal growth factor receptor

The association between angiogenic markers and fetal sex

DEFF Research Database (Denmark)

Andersen, Louise Bjørkholt; Jørgensen, J S; Herse, F

2016-01-01

OBJECTIVE: Current research suggests sexual dimorphism between the male and female fetoplacental units, but with unknown relevance for preeclampsia. We investigated the association between fetal sex and concentrations of the angiogenic markers soluble Fms-like kinase 1 (sFlt-1), placental growth...... factor (PlGF), and sFlt-1/PlGF ratio in first and second-third trimester in women with/without preeclampsia, and the impact of fetal sex on the prognostic value of angiogenic markers for preeclampsia. STUDY DESIGN: Observational study in a prospective, population-based cohort of 2110 singleton...... (preeclampsia cases) associated with fetal sex in adjusted analyses (pfetal sex (all, p=0.028; preeclampsia, p=0.067) In receiver operating curve analysis, prediction of early-onset preeclampsia by sFlt-1/PlGF tended to be superior...

Neurotrophin-3 and FLT3 Tyrosine Kinase Receptor in Perinatal Life

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Ariadne Malamitsi-Puchner

2005-01-01

Full Text Available Our aim is to determine—in 30 healthy full-term infants and their mothers—circulating levels of neurotrophin-3 (NT-3 (important for antenatal and postnatal brain development and implicated in the immune response and FLT3 tyrosine kinase receptor (FLT3 (controlling hematopoiesis and found in the nervous tissue, in the fetal and neonatal life. NT-3 levels, in contrast to FLT3 ones, increased significantly on the fourth postnatal day in relation to the low levels found in the mother, fetus, and day 1 neonate (P=.03, respectively. Maternal and umbilical NT3 levels positively correlated with respective FLT3 levels (P=.003 and P=.03. Circulating NT-3 levels increased in early neonatal life, possibly due to exposure to various stimuli soon after birth. FLT3 levels do not seem to behave accordingly, although these two substances probably synergize.

Novel Indications for Bruton’s Tyrosine Kinase Inhibitors, beyond Hematological Malignancies

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Robert Campbell

2018-03-01

Full Text Available Bruton’s tyrosine kinase (BTK is a critical terminal enzyme in the B-cell antigen receptor (BCR pathway. BTK activation has been implicated in the pathogenesis of certain B-cell malignancies. Targeting this pathway has emerged as a novel target in B-cell malignancies, of which ibrutinib is the first-in-class agent. A few other BTK inhibitors (BTKi are also under development (e.g., acalabrutinib. While the predominant action of BTKi is the blockade of B-cell receptor pathway within malignant B-cells, increasing the knowledge of off-target effects as well as a potential role for B-cells in proliferation of solid malignancies is expanding the indication of BTKi into non-hematological malignancies. In addition to the expansion of the role of BTKi monotherapy, combination therapy strategies utilizing ibrutinib with established regimens and combination with modern immunotherapy compounds are being explored.

Membrane skeletal proteins and their integral membrane protein anchors are targets for tyrosine and threonine kinases in Euglena.

Science.gov (United States)

Fazio, M J; Da Silva, A C; Rosiere, T K; Bouck, G B

1995-01-01

Proteins of the membrane skeleton of Euglena gracilis were extensively phosphorylated in vivo and in vitro after incubation with [32P]-orthophosphate or gamma-[32P] ATP. Endogenous protein threonine/serine activity phosphorylated the major membrane skeletal proteins (articulins) and the putative integral membrane protein (IP39) anchor for articulins. The latter was also the major target for endogenous protein tyrosine kinase activity. A cytoplasmic domain of IP39 was specifically phosphorylated, and removal of this domain with papain eliminated the radiolabeled phosphoamino acids and eliminated or radically shifted the PI of the multiple isoforms of IP39. In gel kinase assays IP39 autophosphorylated and a 25 kDa protein which does not autophosphorylate was identified as a threonine/serine (casein) kinase. Plasma membranes from the membrane skeletal protein complex contained threonine/serine (casein) kinase activity, and cross-linking experiments suggested that IP39 was the likely source for this membrane activity. pH optima, cation requirements and heparin sensitivity of the detergent solubilized membrane activity were determined. Together these results suggest that protein kinases may be important modulators of protein assembly and function of the membrane skeleton of these protistan cells.

Date syrup-derived polyphenols attenuate angiogenic responses and exhibits anti-inflammatory activity mediated by vascular endothelial growth factor and cyclooxygenase-2 expression in endothelial cells.

Science.gov (United States)

Taleb, Hajer; Morris, R Keith; Withycombe, Cathryn E; Maddocks, Sarah E; Kanekanian, Ara D

2016-07-01

Bioactive components such as polyphenols, present in many plants, are purported to have anti-inflammatory and antiangiogenic properties. Date syrup, produced from date fruit of the date palm tree, has traditionally been used to treat a wide range of diseases with etiologies involving angiogenesis and inflammation. It was hypothesized that polyphenols in date syrup reduce angiogenic responses such as cell migration, tube formation, and matrix metalloproteinase activity in an inflammatory model by exhibiting anti-inflammatory activity mediated by vascular endothelial growth factor (VEGF) and the prostaglandin enzyme cyclooxygenase-2 (COX-2) in endothelial cells. Date syrup polyphenols at 60 and 600μg/mL reduced inflammation and suppressed several stages of angiogenesis, including endothelial cell migration, invasion, matrix metalloproteinase activity, and tube formation, without evidence of cytotoxicity. VEGF and COX-2 expression induced by tumor necrosis factor-alpha at both gene expression and protein level was significantly reduced by date syrup polyphenols in comparison to untreated cells. In conclusion, polyphenols in date syrup attenuated angiogenic responses and exhibited anti-inflammatory activity mediated by VEGF and COX-2 expression in endothelial cells. Copyright © 2016 Elsevier Inc. All rights reserved.

The First Pentacyclic Triterpenoid Gypsogenin Derivative Exhibiting Anti-ABL1 Kinase and Anti-chronic Myelogenous Leukemia Activities.

Science.gov (United States)

Ciftci, Halil Ibrahim; Ozturk, Safiye Emirdag; Ali, Taha F S; Radwan, Mohamed O; Tateishi, Hiroshi; Koga, Ryoko; Ocak, Zeynep; Can, Mustafa; Otsuka, Masami; Fujita, Mikako

2018-04-01

The discovery of the chimeric tyrosine kinase breakpoint cluster region kinase-Abelson kinase (BCR-ABL)-targeted drug imatinib conceptually changed the treatment of chronic myelogenous leukemia (CML). However, some CML patients show drug resistance to imatinib. To address this issue, some artificial heterocyclic compounds have been identified as BCR-ABL inhibitors. Here we examined whether plant-derived pentacyclic triterpenoid gypsogenin and/or their derivatives show inhibitory activity against BCR-ABL. Among the three derivatives, benzyl 3-hydroxy-23-oxoolean-12-en-28-oate (1c) was found to be the most effective anticancer agent on the CML cell line K562, with an IC 50 value of 9.3 µM. In contrast, the IC 50 against normal peripheral blood mononuclear cells was 276.0 µM, showing better selectivity than imatinib. Compound 1c had in vitro inhibitory activity against Abelson kinase 1 (ABL1) (IC 50 =8.7 µM), the kinase component of BCR-ABL. In addition, compound 1c showed a different inhibitory profile against eight kinases compared with imatinib. The interaction between ATP binding site of ABL and 1c was examined by molecular docking study, and the binding mode was different from imatinib and newer generation inhibitors. Furthermore, 1c suppressed signaling downstream of BCR-ABL. This study suggests the possibility that plant extracts may be a source for CML treatment and offer a strategy to overcome drug resistance to known BCR-ABL inhibitors.

Application of a drug delivery system using ultrasound and nano/microbubbles for anti-angiogenic therapy

International Nuclear Information System (INIS)

Horie, Sachiko; Kodama, Tetsuya; Sato, Yasushi

2017-01-01

The drug delivery system using ultrasound and nano/microbubbles is a molecular delivery approach using the mechanism of sonoporation. With sonoporation, an endothelium-derived negative-feedback regulator of angiogenesis, Vasohibin-1 (VASH1), was introduced specifically into tumor vessels. We found VASH1 in tumor vessels induce normalization of tumor vessels and inhibited tumor growth. A recent topic regarding tumor angiogenesis is vascular normalization. Tumor vessels are abnormal or immature that cause hyperpermeability and impaired blood flow. Tumor vascular normalization improves blood flow and tissue hypoxia, which increase the effectiveness of chemotherapy and radiotherapy and reduce tumor cell malignancy. In this review, application of drug delivery system using ultrasound for an anti-angiogenic therapy, a tumor vessel normalization therapy to treat cancer, is summarized. (author)

Disabled is a putative adaptor protein that functions during signaling by the sevenless receptor tyrosine kinase.

Science.gov (United States)

Le, N; Simon, M A

1998-08-01

DRK, the Drosophila homolog of the SH2-SH3 domain adaptor protein Grb2, is required during signaling by the sevenless receptor tyrosine kinase (SEV). One role of DRK is to provide a link between activated SEV and the Ras1 activator SOS. We have investigated the possibility that DRK performs other functions by identifying additional DRK-binding proteins. We show that the phosphotyrosine-binding (PTB) domain-containing protein Disabled (DAB) binds to the DRK SH3 domains. DAB is expressed in the ommatidial clusters, and loss of DAB function disrupts ommatidial development. Moreover, reduction of DAB function attenuates signaling by a constitutively activated SEV. Our biochemical analysis suggests that DAB binds SEV directly via its PTB domain, becomes tyrosine phosphorylated upon SEV activation, and then serves as an adaptor protein for SH2 domain-containing proteins. Taken together, these results indicate that DAB is a novel component of the SEV signaling pathway.

Paralog-Specific Patterns of Structural Disorder and Phosphorylation in the Vertebrate SH3-SH2-Tyrosine Kinase Protein Family.

Science.gov (United States)

Dos Santos, Helena G; Siltberg-Liberles, Jessica

2016-09-19

One of the largest multigene families in Metazoa are the tyrosine kinases (TKs). These are important multifunctional proteins that have evolved as dynamic switches that perform tyrosine phosphorylation and other noncatalytic activities regulated by various allosteric mechanisms. TKs interact with each other and with other molecules, ultimately activating and inhibiting different signaling pathways. TKs are implicated in cancer and almost 30 FDA-approved TK inhibitors are available. However, specific binding is a challenge when targeting an active site that has been conserved in multiple protein paralogs for millions of years. A cassette domain (CD) containing SH3-SH2-Tyrosine Kinase domains reoccurs in vertebrate nonreceptor TKs. Although part of the CD function is shared between TKs, it also presents TK specific features. Here, the evolutionary dynamics of sequence, structure, and phosphorylation across the CD in 17 TK paralogs have been investigated in a large-scale study. We establish that TKs often have ortholog-specific structural disorder and phosphorylation patterns, while secondary structure elements, as expected, are highly conserved. Further, domain-specific differences are at play. Notably, we found the catalytic domain to fluctuate more in certain secondary structure elements than the regulatory domains. By elucidating how different properties evolve after gene duplications and which properties are specifically conserved within orthologs, the mechanistic understanding of protein evolution is enriched and regions supposedly critical for functional divergence across paralogs are highlighted. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Next generation metronomic chemotherapy-report from the Fifth Biennial International Metronomic and Anti-angiogenic Therapy Meeting, 6-8 May 2016, Mumbai.

Science.gov (United States)

Pantziarka, Pan; Hutchinson, Lisa; André, Nicolas; Benzekry, Sébastien; Bertolini, Francesco; Bhattacharjee, Atanu; Chiplunkar, Shubhada; Duda, Dan G; Gota, Vikram; Gupta, Sudeep; Joshi, Amit; Kannan, Sadhana; Kerbel, Robert; Kieran, Mark; Palazzo, Antonella; Parikh, Aparna; Pasquier, Eddy; Patil, Vijay; Prabhash, Kumar; Shaked, Yuval; Sholler, Giselle Saulnier; Sterba, Jaroslav; Waxman, David J; Banavali, Shripad

2016-01-01

The 5 th Biennial Metronomic and Anti-angiogenic Therapy Meeting was held on 6 th - 8 th May in the Indian city of Mumbai. The meeting brought together a wide range of clinicians and researchers interested in metronomic chemotherapy, anti-angiogenics, drug repurposing and combinations thereof. Clinical experiences, including many from India, were reported and discussed in three symposia covering breast cancer, head and neck cancers and paediatrics. On the pre-clinical side research into putative mechanisms of action, and the interactions between low dose metronomic chemotherapy and angiogenesis and immune responses, were discussed in a number of presentations. Drug repurposing was discussed both in terms of clinical results, particularly with respect to angiosarcoma and high-risk neuroblastoma, and in pre-clinical settings, particularly the potential for peri-operative interventions. However, it was clear that there remain a number of key areas of challenge, particularly in terms of definitions, perceptions in the wider oncological community, mechanisms of action and predictive biomarkers. While the potential for metronomics and drug repurposing in low and middle income countries remains a key theme, it is clear that there is also considerable potential for clinically relevant improvements in patient outcomes even in high income economies.

Fasting potentiates the anticancer activity of tyrosine kinase inhibitors by strengthening MAPK signaling inhibition

Science.gov (United States)

Caffa, Irene; D'Agostino, Vito; Damonte, Patrizia; Soncini, Debora; Cea, Michele; Monacelli, Fiammetta; Odetti, Patrizio; Ballestrero, Alberto; Provenzani, Alessandro; Longo, Valter D.; Nencioni, Alessio

2015-01-01

Tyrosine kinase inhibitors (TKIs) are now the mainstay of treatment in many types of cancer. However, their benefit is frequently short-lived, mandating the search for safe potentiation strategies. Cycles of fasting enhance the activity of chemo-radiotherapy in preclinical cancer models and dietary approaches based on fasting are currently explored in clinical trials. Whether combining fasting with TKIs is going to be potentially beneficial remains unknown. Here we report that starvation conditions increase the ability of commonly administered TKIs, including erlotinib, gefitinib, lapatinib, crizotinib and regorafenib, to block cancer cell growth, to inhibit the mitogen-activated protein kinase (MAPK) signaling pathway and to strengthen E2F-dependent transcription inhibition. In cancer xenografts models, both TKIs and cycles of fasting slowed tumor growth, but, when combined, these interventions were significantly more effective than either type of treatment alone. In conclusion, cycles of fasting or of specifically designed fasting-mimicking diets should be evaluated in clinical studies as a means to potentiate the activity of TKIs in clinical use. PMID:25909220

JAK kinases are required for the bacterial RNA and poly I:C induced tyrosine phosphorylation of PKR

Science.gov (United States)

Bleiblo, Farag; Michael, Paul; Brabant, Danielle; Ramana, Chilakamarti V; Tai, TC; Saleh, Mazen; Parrillo, Joseph E; Kumar, Anand; Kumar, Aseem

2013-01-01

Discriminating the molecular patterns associated with RNA is central to innate immunity. The protein kinase PKR is a cytosolic sensor involved in the recognition of viral dsRNA and triggering interferon-induced signaling. Here, we identified bacterial RNA as a novel distinct pattern recognized by PKR. We show that the tyrosine phosphorylation of PKR induced by either bacterial RNA or poly I:C is impaired in mutant cells lacking TYK2, JAK1, or JAK2 kinases. PKR was found to be a direct substrate for the activated JAKs. Our results indicated that the double-stranded structures of bacterial RNA are required to fully activate PKR. These results suggest that bacterial RNA signaling is analogous in some respects to that of viral RNA and interferons and may have implications in bacterial immunity. PMID:23236554

β-Catenin is required for intrinsic but not extrinsic BCR-ABL1 kinase-independent resistance to tyrosine kinase inhibitors in chronic myeloid leukemia.

Science.gov (United States)

Eiring, A M; Khorashad, J S; Anderson, D J; Yu, F; Redwine, H M; Mason, C C; Reynolds, K R; Clair, P M; Gantz, K C; Zhang, T Y; Pomicter, A D; Kraft, I L; Bowler, A D; Johnson, K; Partlin, M Mac; O'Hare, T; Deininger, M W

2015-12-01

Activation of nuclear β-catenin and expression of its transcriptional targets promotes chronic myeloid leukemia (CML) progression, tyrosine kinase inhibitor (TKI) resistance, and leukemic stem cell self-renewal. We report that nuclear β-catenin has a role in leukemia cell-intrinsic but not -extrinsic BCR-ABL1 kinase-independent TKI resistance. Upon imatinib inhibition of BCR-ABL1 kinase activity, β-catenin expression was maintained in intrinsically resistant cells grown in suspension culture and sensitive cells cultured in direct contact (DC) with bone marrow (BM) stromal cells. Thus, TKI resistance uncouples β-catenin expression from BCR-ABL1 kinase activity. In β-catenin reporter assays, intrinsically resistant cells showed increased transcriptional activity versus parental TKI-sensitive controls, and this was associated with restored expression of β-catenin target genes. In contrast, DC with BM stromal cells promoted TKI resistance, but had little effects on Lef/Tcf reporter activity and no consistent effects on cytoplasmic β-catenin levels, arguing against a role for β-catenin in extrinsic TKI resistance. N-cadherin or H-cadherin blocking antibodies abrogated DC-based resistance despite increasing Lef/Tcf reporter activity, suggesting that factors other than β-catenin contribute to extrinsic, BM-derived TKI resistance. Our data indicate that, while nuclear β-catenin enhances survival of intrinsically TKI-resistant CML progenitors, it is not required for extrinsic resistance mediated by the BM microenvironment.

Bruton's Tyrosine Kinase: An Emerging Key Player in Innate Immunity.

Science.gov (United States)

Weber, Alexander N R; Bittner, Zsofia; Liu, Xiao; Dang, Truong-Minh; Radsak, Markus Philipp; Brunner, Cornelia

2017-01-01

Bruton's tyrosine kinase (BTK) was initially discovered as a critical mediator of B cell receptor signaling in the development and functioning of adaptive immunity. Growing evidence also suggests multiple roles for BTK in mononuclear cells of the innate immune system, especially in dendritic cells and macrophages. For example, BTK has been shown to function in Toll-like receptor-mediated recognition of infectious agents, cellular maturation and recruitment processes, and Fc receptor signaling. Most recently, BTK was additionally identified as a direct regulator of a key innate inflammatory machinery, the NLRP3 inflammasome. BTK has thus attracted interest not only for gaining a more thorough basic understanding of the human innate immune system but also as a target to therapeutically modulate innate immunity. We here review the latest developments on the role of BTK in mononuclear innate immune cells in mouse versus man, with specific emphasis on the sensing of infectious agents and the induction of inflammation. Therapeutic implications for modulating innate immunity and critical open questions are also discussed.

Reduced intensity conditioning is superior to nonmyeloablative conditioning for older chronic myelogenous leukemia patients undergoing hematopoietic cell transplant during the tyrosine kinase inhibitor era

DEFF Research Database (Denmark)

Warlick, Erica; Ahn, Kwang Woo; Pedersen, Tanya L

2012-01-01

Tyrosine kinase inhibitors (TKIs) and reduced intensity conditioning (RIC)/nonmyeloablative (NMA) conditioning hematopoietic cell transplants (HCTs) have changed the therapeutic strategy for chronic myelogenous leukemia (CML) patients. We analyzed post-HCT outcomes of 306 CML patients reported to...

Pro- and anti-angiogenic factors in human skeletal muscle in response to acute exercise and training

DEFF Research Database (Denmark)

Høier, Birgitte; Nordsborg, Nikolai; Andersen, Søren

2012-01-01

This study examined the effect of acute exercise and 4 weeks of aerobic training on skeletal muscle gene and protein expression of pro- and anti-angiogenic factors in 14 young male subjects. Training consisted of 60 min of cycling (~ 60% of VO2 max), 3 times/week. Biopsies were obtained from m. v....... lateralis before and after training. Muscle interstitial fluid was collected during cycling at week 0 and 4. Training increased (P ... to acute exercise increased similarly (>6-fold; P training. Resting protein levels of soluble VEGF receptor-1 in interstitial fluid, and of VEGF, Thrombospondin-1 (TSP-1) and Tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in muscle, were unaffected by training, whereas e...

Involvement of Src tyrosine kinase and protein kinase C in the expression of macrophage migration inhibitory factor induced by H{sub 2}O{sub 2} in HL-1 mouse cardiac muscle cells

Energy Technology Data Exchange (ETDEWEB)

Rao, F. [Department of Cardiology, Guangdong General Hospital, Guangdong Cardiovascular Institute, Guangdong Academy of Medical Sciences, Guangzhou (China); Research Center of Medical Sciences, Guangdong General Hospital, Guangzhou (China); Guangdong Academy of Medical Sciences, Guangzhou (China); Deng, C.Y. [Research Center of Medical Sciences, Guangdong General Hospital, Guangzhou (China); Guangdong Academy of Medical Sciences, Guangzhou (China); Zhang, Q.H.; Xue, Y.M. [Department of Cardiology, Guangdong General Hospital, Guangdong Cardiovascular Institute, Guangdong Academy of Medical Sciences, Guangzhou (China); Guangdong Academy of Medical Sciences, Guangzhou (China); Xiao, D.Z.; Kuang, S.J.; Lin, Q.X.; Shan, Z.X.; Liu, X.Y.; Zhu, J.N. [Research Center of Medical Sciences, Guangdong General Hospital, Guangzhou (China); Guangdong Academy of Medical Sciences, Guangzhou (China); Yu, X.Y. [Department of Cardiology, Guangdong General Hospital, Guangdong Cardiovascular Institute, Guangdong Academy of Medical Sciences, Guangzhou (China); Research Center of Medical Sciences, Guangdong General Hospital, Guangzhou (China); Guangdong Academy of Medical Sciences, Guangzhou (China); Wu, S.L. [Department of Cardiology, Guangdong General Hospital, Guangdong Cardiovascular Institute, Guangdong Academy of Medical Sciences, Guangzhou (China); Guangdong Academy of Medical Sciences, Guangzhou (China)

2013-09-06

Macrophage migration inhibitory factor (MIF), a pleiotropic cytokine, plays an important role in the pathogenesis of atrial fibrillation; however, the upstream regulation of MIF in atrial myocytes remains unclear. In the present study, we investigated whether and how MIF is regulated in response to the renin-angiotensin system and oxidative stress in atrium myocytes (HL-1 cells). MIF protein and mRNA levels in HL-1 cells were assayed using immunofluorescence, real-time PCR, and Western blot. The result indicated that MIF was expressed in the cytoplasm of HL-1 cells. Hydrogen peroxide (H{sub 2}O{sub 2}), but not angiotensin II, stimulated MIF expression in HL-1 cells. H{sub 2}O{sub 2}-induced MIF protein and gene levels increased in a dose-dependent manner and were completely abolished in the presence of catalase. H{sub 2}O{sub 2}-induced MIF production was completely inhibited by tyrosine kinase inhibitors genistein and PP1, as well as by protein kinase C (PKC) inhibitor GF109203X, suggesting that redox-sensitive MIF production is mediated through tyrosine kinase and PKC-dependent mechanisms in HL-1 cells. These results suggest that MIF is upregulated by HL-1 cells in response to redox stress, probably by the activation of Src and PKC.

Her4 and Her2/neu tyrosine kinase domains dimerize and activate in a reconstituted in vitro system.

Science.gov (United States)

Monsey, John; Shen, Wei; Schlesinger, Paul; Bose, Ron

2010-03-05

Her4 (ErbB-4) and Her2/neu (ErbB-2) are receptor-tyrosine kinases belonging to the epidermal growth factor receptor (EGFR) family. Crystal structures of EGFR and Her4 kinase domains demonstrate kinase dimerization and activation through an allosteric mechanism. The kinase domains form an asymmetric dimer, where the C-lobe surface of one monomer contacts the N-lobe of the other monomer. EGFR kinase dimerization and activation in vitro was previously reported using a nickel-chelating lipid-liposome system, and we now apply this system to all other members of the EGFR family. Polyhistidine-tagged Her4, Her2/neu, and Her3 kinase domains are bound to these nickel-liposomes and are brought to high local concentration, mimicking what happens to full-length receptors in vivo following ligand binding. Addition of nickel-liposomes to Her4 kinase domain results in 40-fold activation in kinase activity and marked enhancement of C-terminal tail autophosphorylation. Activation of Her4 shows a sigmoidal dependence on kinase concentration, consistent with a cooperative process requiring kinase dimerization. Her2/neu kinase activity is also activated by nickel-liposomes, and is increased further by heterodimerization with Her3 or Her4. The ability of Her3 and Her4 to heterodimerize and activate other family members is studied in vitro. Her3 kinase domain readily activates Her2/neu but is a poor activator of Her4, which differs from the prediction made by the asymmetric dimer model. Mutation of Her3 residues (952)ENI(954) to the corresponding sequence in Her4 enhanced the ability of Her3 to activate Her4, demonstrating that sequence differences on the C-lobe surface influence the heterodimerization and activation of ErbB kinase domains.

Leptin’s Pro-Angiogenic Signature in Breast Cancer

International Nuclear Information System (INIS)

Gonzalez-Perez, Ruben Rene; Lanier, Viola; Newman, Gale

2013-01-01

Obesity is linked to increased incidence of breast cancer. The precise causes and mechanisms of these morbid relationships are unknown. Contradictory data on leptin angiogenic actions have been published. However, accumulating evidence would suggest that leptin’s pro-angiogenic effects in cancer play an essential role in the disease. Leptin, the main adipokine secreted by adipose tissue, is also abnormally expressed together with its receptor (OB-R) by breast cancer cells. Leptin induces proliferation and angiogenic differentiation of endothelial cells upregulates VEGF/VEGFR2 and transactivates VEGFR2 independent of VEGF. Leptin induces two angiogenic factors: IL-1 and Notch that can increase VEGF expression. Additionally, leptin induces the secretion and synthesis of proteases and adhesion molecules needed for the development of angiogenesis. Leptin’s paracrine actions can further affect stromal cells and tumor associated macrophages, which express OB-R and secrete VEGF and IL-1, respectively. A complex crosstalk between leptin, Notch and IL-1 (NILCO) that induces VEGF/VEGFR2 is found in breast cancer. Leptin actions in tumor angiogenesis could amplify, be redundant and/or compensatory to VEGF signaling. Current failure of breast cancer anti-angiogenic therapies emphasizes the necessity of targeting the contribution of other pro-angiogenic factors in breast cancer. Leptin’s impact on tumor angiogenesis could be a novel target for breast cancer, especially in obese patients. However, more research is needed to establish the importance of leptin in tumor angiogenesis. This review is focused on updated information on how leptin could contribute to tumor angiogenesis

Learning curve for the management of tyrosine kinase inhibitors as the first line of treatment for patients with metastatic renal cancer.

Science.gov (United States)

Lendínez-Cano, G; Osman García, I; Congregado Ruiz, C B; Conde Sánchez, J M; Medina López, R A

2018-03-07

To analyse the learning curve for the management of tyrosine kinase inhibitors as the first line of treatment for patients with metastatic renal cancer. We evaluated 32 consecutive patients treated in our department for metastatic renal cancer with tyrosine kinase inhibitors (pazopanib or sunitinib) as first-line treatment between September 2012 and November 2015. We retrospectively analysed this sample. We measured the time to the withdrawal of the first-line treatment, the time to progression and overall survival using Kaplan-Meier curves. The learning curve was analysed with the cumulative sum (CUSUM) methodology. In our series, the median time to the withdrawal of the first-line treatment was 11 months (95% CI 4.9-17.1). The mean time to progression was 30.4 months (95% CI 22.7-38.1), and the mean overall survival was 34.9 months (95% CI 27.8-42). By applying the CUSUM methodology, we obtained a graph for the CUSUM value of the time to withdrawal of the first-line treatment (CUSUM TW), observing 3 well-differentiated phases: phase 1 or initial learning phase (1-15), phase 2 (16-26) in which the management of the drug progressively improved and phase 3 (27-32) of maximum experience or mastery of the management of these drugs. The number of treated patients needed to achieve the proper management of these patients was estimated at 15. Despite the limitations of the sample size and follow-up time, we estimated (in 15 patients) the number needed to reach the necessary experience in the management of these patients with tyrosine kinase inhibitors. We observed no relationship between the time to the withdrawal of the first-line treatment for any cause and progression. Copyright © 2018 AEU. Publicado por Elsevier España, S.L.U. All rights reserved.

Combination of interferon-alpha and 5-fluorouracil inhibits endothelial cell growth directly and by regulation of angiogenic factors released by tumor cells

International Nuclear Information System (INIS)

Wada, Hiroshi; Tanemura, Masahiro; Umeshita, Koji; Doki, Yuichiro; Mori, Masaki; Nagano, Hiroaki; Yamamoto, Hirofumi; Noda, Takehiro; Murakami, Masahiro; Kobayashi, Shogo; Marubashi, Shigeru; Eguchi, Hidetoshi; Takeda, Yutaka

2009-01-01

The combination therapy of interferon (IFN)-alpha and 5-fluorouracil (5-FU) improved the prognosis of the patients with hepatocellular carcinoma (HCC). To determine the molecular mechanisms of the anti-tumor and anti-angiogenic effects, we examined the direct anti-proliferative effects on human umbilical vein endothelial cells (HUVEC) and indirect effects by regulating secretion of angiogenic factors from HCC cells. The direct effects on HUVEC were examined by TUNEL, Annexin-V assays and cell cycles analysis. For analysis of the indirect effects, the apoptosis induced by the conditioned medium from HCC cell treated by IFN-alpha/5-FU and expression of angiogenic factors was examined. IFN-alpha and 5-FU alone had anti-proliferative properties on HUVEC and their combination significantly inhibited the growth (compared with control, 5-FU or IFN alone). TUNEL and Annexin-V assays showed no apoptosis. Cell cycle analysis revealed that IFN-alpha and 5-FU delayed cell cycle progression in HUVEC with S-phase accumulation. The conditioned medium from HuH-7 cells after treatment with IFN/5-FU significantly inhibited HUVEC growth and induced apoptosis, and contained high levels of angiopoietin (Ang)-1 and low levels of vascular endothelial growth factor (VEGF) and Ang-2. Knockdown of Ang-1 in HuH-7 cells abrogated the anti-proliferative effects on HUVEC while knockdown of Ang-2 partially rescue the cells. These results suggested that IFN-alpha and 5-FU had direct growth inhibitory effects on endothelial cells, as well as anti-angiogenic effects through regulation of angiogenic factors released from HCC cells. Modulation of VEGF and Angs secretion by IFN-alpha and 5-FU may contribute to their anti-angiogenic and anti-tumor effects on HCC

Egress of CD19(+)CD5(+) cells into peripheral blood following treatment with the Bruton tyrosine kinase inhibitor ibrutinib in mantle cell lymphoma patients

NARCIS (Netherlands)

Chang, Betty Y.; Francesco, Michelle; de Rooij, Martin F. M.; Magadala, Padmaja; Steggerda, Susanne M.; Huang, Min Mei; Kuil, Annemieke; Herman, Sarah E. M.; Chang, Stella; Pals, Steven T.; Wilson, Wyndham; Wiestner, Adrian; Spaargaren, Marcel; Buggy, Joseph J.; Elias, Laurence

2013-01-01

Ibrutinib (PCI-32765) is a highly potent oral Bruton tyrosine kinase (BTK) inhibitor in clinical development for treating B-cell lymphoproliferative diseases. Patients with chronic lymphocytic leukemia (CLL) often show marked, transient increases of circulating CLL cells following ibrutinib

Systematic characterization of the specificity of the SH2 domains of cytoplasmic tyrosine kinases.

Science.gov (United States)

Zhao, Bing; Tan, Pauline H; Li, Shawn S C; Pei, Dehua

2013-04-09

Cytoplasmic tyrosine kinases (CTK) generally contain a Src-homology 2 (SH2) domain, whose role in the CTK family is not fully understood. Here we report the determination of the specificity of 25 CTK SH2 domains by screening one-bead-one-compound (OBOC) peptide libraries. Based on the peptide sequences selected by the SH2 domains, we built Support Vector Machine (SVM) models for the prediction of binding ligands for the SH2 domains. These models yielded support for the progressive phosphorylation model for CTKs in which the overlapping specificity of the CTK SH2 and kinase domains has been proposed to facilitate targeting of the CTK substrates with at least two potential phosphotyrosine (pTyr) sites. We curated 93 CTK substrates with at least two pTyr sites catalyzed by the same CTK, and showed that 71% of these substrates had at least two pTyr sites predicted to bind a common CTK SH2 domain. More importantly, we found 34 instances where there was at least one pTyr site predicted to be recognized by the SH2 domain of the same CTK, suggesting that the SH2 and kinase domains of the CTKs may cooperate to achieve progressive phosphorylation of a protein substrate. This article is part of a Special Issue entitled: From protein structures to clinical applications. Copyright © 2012 Elsevier B.V. All rights reserved.

Cancer Cell-derived Exosomes Induce Mitogen-activated Protein Kinase-dependent Monocyte Survival by Transport of Functional Receptor Tyrosine Kinases*

Science.gov (United States)

Song, Xiao; Ding, Yanping; Liu, Gang; Yang, Xiao; Zhao, Ruifang; Zhang, Yinlong; Zhao, Xiao; Anderson, Gregory J.; Nie, Guangjun

2016-01-01

Tumor-associated macrophages (TAM) play pivotal roles in cancer initiation and progression. Monocytes, the precursors of TAMs, normally undergo spontaneous apoptosis within 2 days, but can subsist in the inflammatory tumor microenvironment for continuous survival and generation of sufficient TAMs. The mechanisms underlying tumor-driving monocyte survival remain obscure. Here we report that cancer cell-derived exosomes were crucial mediators for monocyte survival in the inflammatory niche. Analysis of the survival-promoting molecules in monocytes revealed that cancer cell-derived exosomes activated Ras and extracellular signal-regulated kinases in the mitogen-activated protein kinase (MAPK) pathway, resulting in the prevention of caspase cleavage. Phosphorylated receptor tyrosine kinases (RTKs), such as phosphorylated epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2), were abundantly expressed in cancer cell-derived exosomes. Knock-out of EGFR or/and HER-2, or alternatively, inhibitors against their phosphorylation significantly disturbed the exosome-mediated activation of the MAPK pathway, inhibition of caspase cleavage, and increase in survival rate in monocytes. Moreover, the deprived survival-stimulating activity of exosomes due to null expression of EGFR and HER-2 could be restored by activation of another RTK, insulin receptor. Overall, our study uncovered a mechanism of tumor-associated monocyte survival and demonstrated that cancer cell-derived exosomes can stimulate the MAPK pathway in monocytes through transport of functional RTKs, leading to inactivation of apoptosis-related caspases. This work provides insights into the long sought question on monocyte survival prior to formation of plentiful TAMs in the tumor microenvironment. PMID:26895960

Identification of nuclear protein targets for six leukemogenic tyrosine kinases governed by post-translational regulation.

Directory of Open Access Journals (Sweden)

Andrew Pierce

Full Text Available Mutated tyrosine kinases are associated with a number of different haematological malignancies including myeloproliferative disorders, lymphoma and acute myeloid leukaemia. The potential commonalities in the action of six of these leukemogenic proteins on nuclear proteins were investigated using systematic proteomic analysis. The effects on over 3600 nuclear proteins and 1500 phosphopeptide sites were relatively quantified in seven isogenic cell lines. The effects of the kinases were diverse although some commonalities were found. Comparison of the nuclear proteomic data with transcriptome data and cytoplasmic proteomic data indicated that the major changes are due to post-translational mechanisms rather than changes in mRNA or protein distribution. Analysis of the promoter regions of genes whose protein levels changed in response to the kinases showed the most common binding site found was that for NFκB whilst other sites such as those for the glucocorticoid receptor were also found. Glucocorticoid receptor levels and phosphorylation were decreased by all 6 PTKs. Whilst Glucocorticoid receptor action can potentiate NFκB action those proteins where genes have NFκB binding sites were in often regulated post-translationally. However all 6 PTKs showed evidence of NFkB pathway modulation via activation via altered IkB and NFKB levels. Validation of a common change was also undertaken with PMS2, a DNA mismatch repair protein. PMS2 nuclear levels were decreased in response to the expression of all 6 kinases, with no concomitant change in mRNA level or cytosolic protein level. Response to thioguanine, that requires the mismatch repair pathway, was modulated by all 6 oncogenic kinases. In summary common targets for 6 oncogenic PTKs have been found that are regulated by post-translational mechanisms. They represent potential new avenues for therapies but also demonstrate the post-translational regulation is a key target of leukaemogenic kinases.

Comparison of the crystal structures of the potent anticancer and anti-angiogenic agent regorafenib and its monohydrate.

Science.gov (United States)

Sun, Meng Ying; Wu, Su Xiang; Zhou, Xin Bo; Gu, Jian Ming; Hu, Xiu Rong

2016-04-01

Regorafenib {systematic name: 4-[4-({[4-chloro-3-(trifluoromethy)phenyl]carbamoyl}amino)-3-fluorophenoxy]-1-methylpyridine-2-carboxamide}, C21H15ClF4N4O3, is a potent anticancer and anti-angiogenic agent that possesses various activities on the VEGFR, PDGFR, raf and/or flt-3 kinase signaling molecules. The compound has been crystallized as polymorphic form I and as the monohydrate, C21H15ClF4N4O3·H2O. The regorafenib molecule consists of biarylurea and pyridine-2-carboxamide units linked by an ether group. A comparison of both forms shows that they differ in the relative orientation of the biarylurea and pyridine-2-carboxamide units, due to different rotations around the ether group, as measured by the C-O-C bond angles [119.5 (3)° in regorafenib and 116.10 (15)° in the monohydrate]. Meanwhile, the conformational differences are reflected in different hydrogen-bond networks. Polymorphic form I contains two intermolecular N-H...O hydrogen bonds, which link the regorafenib molecules into an infinite molecular chain along the b axis. In the monohydrate, the presence of the solvent water molecule results in more abundant hydrogen bonds. The water molecules act as donors and acceptors, forming N-H...O and O-H...O hydrogen-bond interactions. Thus, R4(2)(28) ring motifs are formed, which are fused to form continuous spiral ring motifs along the a axis. The (trifluoromethyl)phenyl rings protrude on the outside of these motifs and interdigitate with those of adjacent ring motifs, thereby forming columns populated by halogen atoms.

P-loop conformation governed crizotinib resistance in G2032R-mutated ROS1 tyrosine kinase: clues from free energy landscape.

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Huiyong Sun

2014-07-01

Full Text Available Tyrosine kinases are regarded as excellent targets for chemical drug therapy of carcinomas. However, under strong purifying selection, drug resistance usually occurs in the cancer cells within a short term. Many cases of drug resistance have been found to be associated with secondary mutations in drug target, which lead to the attenuated drug-target interactions. For example, recently, an acquired secondary mutation, G2032R, has been detected in the drug target, ROS1 tyrosine kinase, from a crizotinib-resistant patient, who responded poorly to crizotinib within a very short therapeutic term. It was supposed that the mutation was located at the solvent front and might hinder the drug binding. However, a different fact could be uncovered by the simulations reported in this study. Here, free energy surfaces were characterized by the drug-target distance and the phosphate-binding loop (P-loop conformational change of the crizotinib-ROS1 complex through advanced molecular dynamics techniques, and it was revealed that the more rigid P-loop region in the G2032R-mutated ROS1 was primarily responsible for the crizotinib resistance, which on one hand, impaired the binding of crizotinib directly, and on the other hand, shortened the residence time induced by the flattened free energy surface. Therefore, both of the binding affinity and the drug residence time should be emphasized in rational drug design to overcome the kinase resistance.

Multisite tyrosine phosphorylation of the N-terminus of Mint1/X11α by Src kinase regulates the trafficking of amyloid precursor protein.

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Dunning, Christopher J R; Black, Hannah L; Andrews, Katie L; Davenport, Elizabeth C; Conboy, Michael; Chawla, Sangeeta; Dowle, Adam A; Ashford, David; Thomas, Jerry R; Evans, Gareth J O

2016-05-01

Mint/X11 is one of the four neuronal trafficking adaptors that interact with amyloid precursor protein (APP) and are linked with its cleavage to generate β-amyloid peptide, a key player in the pathology of Alzheimer's disease. How APP switches between adaptors at different stages of the secretory pathway is poorly understood. Here, we show that tyrosine phosphorylation of Mint1 regulates the destination of APP. A canonical SH2-binding motif ((202) YEEI) was identified in the N-terminus of Mint1 that is phosphorylated on tyrosine by C-Src and recruits the active kinase for sequential phosphorylation of further tyrosines (Y191 and Y187). A single Y202F mutation in the Mint1 N-terminus inhibits C-Src binding and tyrosine phosphorylation. Previous studies observed that co-expression of wild-type Mint1 and APP causes accumulation of APP in the trans-Golgi. Unphosphorylatable Mint1 (Y202F) or pharmacological inhibition of Src reduced the accumulation of APP in the trans-Golgi of heterologous cells. A similar result was observed in cultured rat hippocampal neurons where Mint1(Y202F) permitted the trafficking of APP to more distal neurites than the wild-type protein. These data underline the importance of the tyrosine phosphorylation of Mint1 as a critical switch for determining the destination of APP. The regulation of amyloid precursor protein (APP) trafficking is poorly understood. We have discovered that the APP adapter, Mint1, is phosphorylated by C-Src kinase. Mint1 causes APP accumulation in the trans-Golgi network, whereas inhibition of Src or mutation of Mint1-Y202 permits APP recycling. The phosphorylation status of Mint1 could impact on the pathological trafficking of APP in Alzheimer's disease. © 2016 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.

Receptor tyrosine phosphatase R-PTP-alpha is tyrosine-phosphorylated and associated with the adaptor protein Grb2

DEFF Research Database (Denmark)

Su, J; Batzer, A; Sap, J

1994-01-01

Receptor tyrosine phosphatases (R-PTPases) have generated interest because of their suspected involvement in cellular signal transduction. The adaptor protein Grb2 has been implicated in coupling receptor tyrosine kinases to Ras. We report that a ubiquitous R-PTPase, R-PTP-alpha, is tyrosine......-phosphorylated and associated in vivo with the Grb2 protein. This association can be reproduced in stably and transiently transfected cells, as well as in vitro using recombinant Grb2 protein. Association requires the presence of an intact SH2 domain in Grb2, as well as tyrosine phosphorylation of R-PTP-alpha. This observation...... links a receptor tyrosine phosphatase with a key component of a central cellular signalling pathway and provides a basis for addressing R-PTP-alpha function....

Tyrosine 402 Phosphorylation of Pyk2 Is Involved in Ionomycin-Induced Neurotransmitter Release

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Zhang, Zhao; Zhang, Yun; Mou, Zheng; Chu, Shifeng; Chen, Xiaoyu; He, Wenbin; Guo, Xiaofeng; Yuan, Yuhe; Takahashi, Masami; Chen, Naihong

2014-01-01

Protein tyrosine kinases, which are highly expressed in the central nervous system, are implicated in many neural processes. However, the relationship between protein tyrosine kinases and neurotransmitter release remains unknown. In this study, we found that ionomycin, a Ca2+ ionophore, concurrently induced asynchronous neurotransmitter release and phosphorylation of a non-receptor protein tyrosine kinase, proline-rich tyrosine kinase 2 (Pyk2), in clonal rat pheochromocytoma PC12 cells and cerebellar granule cells, whereas introduction of Pyk2 siRNA dramatically suppressed ionomycin-induced neurotransmitter release. Further study indicated that Tyr-402 (Y402) in Pyk2, instead of other tyrosine sites, underwent rapid phosphorylation after ionomycin induction in 1 min to 2 min. We demonstrated that the mutant of Pyk2 Y402 could abolish ionomycin-induced dopamine (DA) release by transfecting cells with recombinant Pyk2 and its mutants (Y402F, Y579F, Y580F, and Y881F). In addition, Src inhibition could prolong phosphorylation of Pyk2 Y402 and increase DA release. These findings suggested that Pyk2 was involved in ionomycin-induced neurotransmitter release through phosphorylation of Y402. PMID:24718602

Anti-EGFR Therapy: Mechanism and Advances in Clinical Efficacy in Breast Cancer

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John F. Flynn

2009-01-01

Full Text Available This review will focus on recent advances in the application of antiepidermal growth factor receptor (anti-EGFR for the treatment of breast cancer. The choice of EGFR, a member of the ErbB tyrosine kinase receptor family, stems from evidence pinpointing its role in various anti-EGFR therapies. Therefore, an increase in our understanding of EGFR mechanism and signaling might reveal novel targets amenable to intervention in the clinic. This knowledge base might also improve existing medical treatment options and identify research gaps in the design of new therapeutic agents. While the approved use of drugs like the dual kinase inhibitor Lapatinib represents significant advances in the clinical management of breast cancer, confirmatory studies must be considered to foster the use of anti-EGFR therapies including safety, pharmacokinetics, and clinical efficacy.

The SH2 domain of Abl kinases regulates kinase autophosphorylation by controlling activation loop accessibility

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Lamontanara, Allan Joaquim; Georgeon, Sandrine; Tria, Giancarlo; Svergun, Dmitri I.; Hantschel, Oliver

2014-11-01

The activity of protein kinases is regulated by multiple molecular mechanisms, and their disruption is a common driver of oncogenesis. A central and almost universal control element of protein kinase activity is the activation loop that utilizes both conformation and phosphorylation status to determine substrate access. In this study, we use recombinant Abl tyrosine kinases and conformation-specific kinase inhibitors to quantitatively analyse structural changes that occur after Abl activation. Allosteric SH2-kinase domain interactions were previously shown to be essential for the leukemogenesis caused by the Bcr-Abl oncoprotein. We find that these allosteric interactions switch the Abl activation loop from a closed to a fully open conformation. This enables the trans-autophosphorylation of the activation loop and requires prior phosphorylation of the SH2-kinase linker. Disruption of the SH2-kinase interaction abolishes activation loop phosphorylation. Our analysis provides a molecular mechanism for the SH2 domain-dependent activation of Abl that may also regulate other tyrosine kinases.

A Dual Role for the Nonreceptor Tyrosine Kinase Pyk2 during the Intracellular Trafficking of Human Papillomavirus 16.

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Gottschalk, Elinor Y; Meneses, Patricio I

2015-09-01

The infectious process of human papillomaviruses (HPVs) has been studied considerably, and many cellular components required for viral entry and trafficking continue to be revealed. In this study, we investigated the role of the nonreceptor tyrosine kinase Pyk2 during HPV16 pseudovirion infection of human keratinocytes. We found that Pyk2 is necessary for infection and appears to be involved in the intracellular trafficking of the virus. Small interfering RNA-mediated reduction of Pyk2 resulted in a significant decrease in infection but did not prevent viral entry at the plasma membrane. Pyk2 depletion resulted in altered endolysosomal trafficking of HPV16 and accelerated unfolding of the viral capsid. Furthermore, we observed retention of the HPV16 pseudogenome in the trans-Golgi network (TGN) in Pyk2-depleted cells, suggesting that the kinase could be required for the viral DNA to exit the TGN. While Pyk2 has previously been shown to function during the entry of enveloped viruses at the plasma membrane, the kinase has not yet been implicated in the intracellular trafficking of a nonenveloped virus such as HPV. Additionally, these data enrich the current literature on Pyk2's function in human keratinocytes. In this study, we investigated the role of the nonreceptor tyrosine kinase Pyk2 during human papillomavirus (HPV) infection of human skin cells. Infections with high-risk types of HPV such as HPV16 are the leading cause of cervical cancer and a major cause of genital and oropharyngeal cancer. As a nonenveloped virus, HPV enters cells by interacting with cellular receptors and established cellular trafficking routes to ensure that the viral DNA reaches the nucleus for productive infection. This study identified Pyk2 as a cellular component required for the intracellular trafficking of HPV16 during infection. Understanding the infectious pathways of HPVs is critical for developing additional preventive therapies. Furthermore, this study advances our knowledge of

Tyrosine receptor kinase B receptor activation reverses the impairing effects of acute nicotine on contextual fear extinction.

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Kutlu, Munir Gunes; Cole, Robert D; Connor, David A; Natwora, Brendan; Gould, Thomas J

2018-03-01

Anxiety and stress disorders have been linked to deficits in fear extinction. Our laboratory and others have demonstrated that acute nicotine impairs contextual fear extinction, suggesting that nicotine exposure may have negative effects on anxiety and stress disorder symptomatology. However, the neurobiological mechanisms underlying the acute nicotine-induced impairment of contextual fear extinction are unknown. Therefore, based on the previous studies showing that brain-derived neurotrophic factor is central for fear extinction learning and acute nicotine dysregulates brain-derived neurotrophic factor signaling, we hypothesized that the nicotine-induced impairment of contextual fear extinction may involve changes in tyrosine receptor kinase B signaling. To test this hypothesis, we systemically, intraperitoneally, injected C57BL/6J mice sub-threshold doses (2.5 and 4.0 mg/kg) of 7,8-dihydroxyflavone, a small-molecule tyrosine receptor kinase B agonist that fully mimics the effects of brain-derived neurotrophic factor, or vehicle an hour before each contextual fear extinction session. Mice also received injections, intraperitoneally, of acute nicotine (0.18 mg/kg) or saline 2-4 min before extinction sessions. While the animals that received only 7,8-dihydroxyflavone did not show any changes in contextual fear extinction, 4.0 mg/kg of 7,8-dihydroxyflavone ameliorated the extinction deficits in mice administered acute nicotine. Overall, these results suggest that acute nicotine-induced impairment of context extinction may be related to a disrupted brain-derived neurotrophic factor signaling.

A receptor tyrosine kinase inhibitor, Tyrphostin A9 induces cancer cell death through Drp1 dependent mitochondria fragmentation

International Nuclear Information System (INIS)

Park, So Jung; Park, Young Jun; Shin, Ji Hyun; Kim, Eun Sung; Hwang, Jung Jin; Jin, Dong-Hoon; Kim, Jin Cheon; Cho, Dong-Hyung

2011-01-01

Highlights: → We screened and identified Tyrphostin A9, a receptor tyrosine kinase inhibitor as a strong mitochondria fission inducer. → Tyrphostin A9 treatment promotes mitochondria dysfunction and contributes to cytotoxicity in cancer cells. → Tyrphostin A9 induces apoptotic cell death through a Drp1-mediated pathway. → Our studies suggest that Tyrphostin A9 induces mitochondria fragmentation and apoptotic cell death via Drp1 dependently. -- Abstract: Mitochondria dynamics controls not only their morphology but also functions of mitochondria. Therefore, an imbalance of the dynamics eventually leads to mitochondria disruption and cell death. To identify specific regulators of mitochondria dynamics, we screened a bioactive chemical compound library and selected Tyrphostin A9, a tyrosine kinase inhibitor, as a potent inducer of mitochondrial fission. Tyrphostin A9 treatment resulted in the formation of fragmented mitochondria filament. In addition, cellular ATP level was decreased and the mitochondrial membrane potential was collapsed in Tyr A9-treated cells. Suppression of Drp1 activity by siRNA or over-expression of a dominant negative mutant of Drp1 inhibited both mitochondrial fragmentation and cell death induced by Tyrpohotin A9. Moreover, treatment of Tyrphostin A9 also evoked mitochondrial fragmentation in other cells including the neuroblastomas. Taken together, these results suggest that Tyrphostin A9 induces Drp1-mediated mitochondrial fission and apoptotic cell death.

Immunotoxicity assessment for the novel Spleen tyrosine kinase inhibitor R406

International Nuclear Information System (INIS)

Zhu Yanhong; Herlaar, Ellen; Masuda, Esteban S.; Burleson, Gary R.; Nelson, Andrew J.; Grossbard, Elliott B.; Clemens, George R.

2007-01-01

Spleen tyrosine kinase (Syk) is a novel pharmaceutical target for treatment of allergic, autoimmune, and neoplastic disorders. Previous studies have indicated that Syk signaling plays critical roles in regulating the lymphohematopoietic system. These observations prompted us to investigate whether inhibition of Syk would promote immunotoxicity. In a series of studies, rats were treated orally with R406, at dose levels up to and including 100 mg/kg/day (or its prodrug R788 at dose levels up to and including 100 mg/kg/day, reduced to 50 mg/kg/day for females as MTD was exceeded), a potent Syk inhibitor, twice daily for 28 days. In addition to standard toxicological assessments, immunophenotyping by flow cytometric analysis, and a study of humoral immune response measuring anti-KLH IgM and IgG levels, were undertaken. Other immunotoxicity studies included three host resistance models in female Balb/c mice to further ascertain effects of R406 on innate and acquired immunity. Following R406 treatment, expected immunomodulating effects (e.g., decreased thymic and spleen weight, hypocellularity of bone marrow, and reduced lymphocyte counts, including T and B cells) were observed in the rat studies. These changes essentially resolved during a 14-day treatment-free recovery period. A KLH challenge in rats demonstrated no adverse effects on IgG or IgM response. R788/406, administered orally at dose levels up to and including 80 mg/kg/day for 28 days, did not affect bacterial or viral clearance in the Listeria, Streptococcal, or Influenza host resistance mouse models, respectively. This correlated with previous in vitro macrophage and neutrophil function assays (assessing migration, phagocytosis, oxidative burst and microbicidal activity), which revealed that R406 did not adversely affect macrophage or neutrophil function in innate immune responses. Collectively, these results demonstrate that R406 has minimal functional immunotoxicity notwithstanding its lymphocytopenic

Tyrosine dephosphorylation enhances the therapeutic target activity of epidermal growth factor receptor (EGFR) by disrupting its interaction with estrogen receptor (ER).

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Ma, Shao; Yin, Ning; Qi, Xiaomei; Pfister, Sandra L; Zhang, Mei-Jie; Ma, Rong; Chen, Guan

2015-05-30

Protein-protein interactions can increase or decrease its therapeutic target activity and the determining factors involved, however, are largely unknown. Here, we report that tyrosine-dephosphorylation of epidermal growth factor receptor (EGFR) increases its therapeutic target activity by disrupting its interaction with estrogen receptor (ER). Protein tyrosine phosphatase H1 (PTPH1) dephosphorylates the tyrosine kinase EGFR, disrupts its interaction with the nuclear receptor ER, and increases breast cancer sensitivity to small molecule tyrosine kinase inhibitors (TKIs). These effects require PTPH1 catalytic activity and its interaction with EGFR, suggesting that the phosphatase may increase the sensitivity by dephosphorylating EGFR leading to its dissociation with ER. Consistent with this notion, a nuclear-localization defective ER has a higher EGFR-binding activity and confers the resistance to TKI-induced growth inhibition. Additional analysis show that PTPH1 stabilizes EGFR, stimulates the membranous EGFR accumulation, and enhances the growth-inhibitory activity of a combination therapy of TKIs with an anti-estrogen. Since EGFR and ER both are substrates for PTPH1 in vitro and in intact cells, these results indicate that an inhibitory EGFR-ER protein complex can be switched off through a competitive enzyme-substrate binding. Our results would have important implications for the treatment of breast cancer with targeted therapeutics.

Tyrosine kinase inhibitors directed against the vascular endothelial growth factor receptor (VEGFR) have distinct cutaneous toxicity profiles: a meta-analysis and review of the literature.

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Massey, Paul R; Okman, Jonathan S; Wilkerson, Julia; Cowen, Edward W

2015-06-01

Inhibition of the vascular endothelial growth factor receptor (VEGFR) with tyrosine kinase inhibitors (TKIs) is associated with cutaneous adverse effects that increase patient morbidity. Our objective was to examine the skin toxicity profile of anti-VEGFR TKIs and determine the changing incidence in clinical trials. PubMed was queried for phase II or III trials of anti-VEGFR TKIs between 2000 and 2013 involving ≥50 patients. Adverse events were abstracted, with results presented in both fixed and random effects models. Odds ratios (OR) and 95 % confidence intervals (CIs) were estimated for studies with at least two arms. Across 82 included studies, all grades rash (OR, 2.68; 95 % CI, 2.45-2.94), hand-foot skin reaction (HFSR) (OR, 2.70; 95 % CI, 2.43-3.00), and pruritus (OR, 1.25; 95 % CI, 1.12-1.39) were associated with anti-VEGFR TKIs. Vandetanib had the highest incidence of rash (41 %), while sorafenib was most commonly associated with HFSR (37 %) and pruritus (14 %). The incidence of HFSR from 2000 to 2013 showed an upward trend (r (2) = 0.042, p = 0.10) and in sunitinib therapy increased significantly (r (2) = 0.237, p = 0.04). The incidence of HFSR, rash, and pruritus varies considerably by drug. Our data suggest a continued need to address skin toxicities and improve reporting strategies.

The RON receptor tyrosine kinase in pancreatic cancer pathogenesis and its potential implications for future targeted therapies.

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Kang, Chang Moo; Babicky, Michele L; Lowy, Andrew M

2014-03-01

Pancreatic cancer remains a devastating disease with a mortality rate that has not changed substantially in decades. Novel therapies are therefore desperately needed. The RON receptor tyrosine kinase has been identified as an important mediator of KRAS oncogene addiction and is overexpressed in the majority of pancreatic cancers. Preclinical studies show that inhibition of RON function decreases pancreatic cancer cell migration, invasion, and survival and can sensitize pancreatic cancer cells to chemotherapy. This article reviews the current state of knowledge regarding RON biology and pancreatic cancer and discusses its potential as a therapeutic target.

Antibody-mediated platelet phagocytosis by human macrophages is inhibited by siRNA specific for sequences in the SH2 tyrosine kinase, Syk.

Science.gov (United States)

Lu, Ying; Wang, Weiming; Mao, Huiming; Hu, Hai; Wu, Yanling; Chen, Bing-Guan; Liu, Zhongmin

2011-01-01

Immune thrombocytopenia depends upon Fc receptor-mediated phagocytosis that involves signaling through the SH2 tyrosine kinase, Syk. We designed small interfering (siRNA) sequences complementary to Syk coding regions to decrease the expression of Syk in the human macrophage cell line, THP-1. To evaluate the functional effect of siRNA on phagocytosis, we developed a new in vitro assay for antibody-mediated platelet ingestion by THP-1 cells. Incubation of THP-1 cells at 37°C with fluorescence-labeled platelets and anti-platelet antibody promoted ingestion of platelets that could be quantitated by flow cytometry. Transfection of THP-1 cells with Syk-specific siRNA resulted in a reduction in the amount of FcγRII-associated Syk protein. Coincident with decreased Syk expression, we observed inhibition of antibody-mediated platelet ingestion. These results confirm a key role for Syk in antibody-mediated phagocytosis and suggest Syk-specific siRNA as a possible therapeutic candidate for immune thrombocytopenia. Copyright © 2011 Elsevier Inc. All rights reserved.

Liquid chromatography-tandem mass spectrometric assay for the quantitative determination of the tyrosine kinase inhibitor quizartinib in mouse plasma using salting-out liquid-liquid extraction