bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 3879: High-throughput, homogeneous in cyto assays to monitor histone H3 post-translational modifications
Authors: Beaudet Lucille , Sergerie Jean-Philippe Lévesque , Boulé Marie , Labonté Anne , Michaud Jean-François , Rouleau Nathalie , Arcand Mathieu 
Published: August 2012

Abstract
Histone proteins are an integral part of DNA packaging into chromatin, a dynamic process partly regulated by post-translation modification of histone N-terminal tails. Aberrant histone acetylation (ac) or methylation (me) levels is associated with a number of diseases. For example, histone deacetylases (HDACs and sirtuins) regulate the expression of many genes involved in neurodegeneration and can be aberrantly expressed in different tumors. In addition, deregulation of the methyl marks H3K4me3 and H3K27me3 are found associated with the development of several types of cancer. This work presents the development of high-throughput AlphaLISA® in cyto assays to monitor four specific histone marks: H3K9ac, H3K4me2, H3K27ac and H3K27me3. These assays were performed using an all-in-one well histone extraction protocol requiring no acid extraction or centrifugation steps. The level of each histone mark was first measured in cell titration experiments, seeding cells from 500 to 10 000 in 384 well plates. The chemical modulation of H3K4me2, H3K9ac and H3K27ac was monitored following overnight treatment of HeLa, HEK293, and Jurkat cells with the non-selective histone deacetylase inhibitors sodium butyrate and trichostatin A. Signal increases were all corroborated by Western blot analysis using the same antibodies as in the AlphaLISA detection assay. In the absence of chemical modulation, measurement of different H3K27me3 mark levels was performed in two B cell lymphoma cell lines: OCI-LY-19 and SU-DHL-6. OCI-LY-19 cells express wild-type EZH2 methyltransferase while SU-DHL-6 cells bear a heterozygous EZH2 mutation (Y641N) that alters its substrate selectivity, resulting in increased H3K27me3 levels. These novel cell-based assays showed suitability for high-throughput screening (HTS) protocols as demonstrated by Z’ factors superior to 0.6 for all four marks. In summary, these homogeneous cell-based AlphaLISA assays allow the simple and rapid monitoring of cellular histone H3 mark levels.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3879. doi:1538-7445.AM2012-3879
Abstract 3880: Application of tyramide signal amplification technology to high content analysis of cellular kinase translocation assays
Authors: Hurt Stephen , Campisano Michael , Letzsch Stefan 
Published: August 2012

Abstract
Deregulation of cellular kinase signaling pathways plays a key role in the ability of cancer cells to sustain uncontrolled proliferation, resist apoptosis, and undergo metastasis. The activation of kinase transduction pathways commonly involves the translocation of various molecular components of the pathway from one subcellular compartment to another. For example, growth factor stimulation of cells activates signaling pathways that are regulated by the level of phosphorylation of protein kinases such as protein kinase C (PKC) and protein kinase B (PKB/Akt); PKC and Akt are first recruited from the cytosol to the cell membrane where the phosphorylation event occurs. High Content Analysis (HCA) assays have become important tools in screening for potential new drugs to modulate aberrant signaling pathways. Confocal imaging provides the highest degree of spatial resolution, and can also yield lower background signals than wide-field imaging. The downside of confocal imaging is that it requires longer signal acquisition times, and it may be difficult to detect targets present in low abundance. The longer acquisition time can also result in decreased throughput when HCA is used as the detection mode for high throughput screening. Tyramide signal amplification (TSA) is an enzyme-based technology that has previous been used to enhance signal sensitivity in immunochemical staining procedures. We have applied TSA technology to the HCA of cellular kinase translocation assays using the Operetta High Content Imaging System in confocal fluorescence mode. HeLa cells were treated with compounds that induced kinase cytosol to plasma membrane translocation. Kinase localization was measured using a texture-based image analysis algorithm. We compared the signal intensity with and without TSA amplification. We will present data showing that TSA enhanced the signal intensity by at least an order of magnitude without a loss of resolution. As a result, the detection antibody can be reduced 5-fold, and the exposure time reduced 10-fold. In conclusion, TSA amplification of HCA kinase translocation assays can significantly increase sample throughput when screening compound libraries.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3880. doi:1538-7445.AM2012-3880
Abstract 3881: BacMam-enabled biochemical and cellular assays to assess inhibitors of full-length receptor tyrosine kinases
Authors: Hallis Tina , Lebakken Connie , Reichling Laurie , Ellefson Jason , Carlson Coby , Hermanson Spencer , Bi Kun , Riddle Steve 
Published: August 2012

Abstract
Receptor tyrosine kinases (RTKs) are important pharmacological targets in oncology. Current biochemical assays designed to assess compound efficacy utilize truncated forms of the kinase lacking the transmembrane or extracellular domains and fail to address how these domains might affect the observed pharmacology. Purification of the full-length recombinant receptor can be both costly and difficult to scale for large screening campaigns. Additionally, because of the generic nature of most substrates used in activity assays for RTKs, small amounts of contaminating kinases can interfere with assay results. Numerous cell-based assays for pathway analysis can also serve as readouts for these targets, but they are not a direct measurement of kinase activity. To address these limitations, we have employed a BacMam-mediated gene delivery system to express full-length receptor tyrosine kinases with C-terminal green fluorescent protein (GFP) fusions in a variety of cellular backgrounds. Expression of these full-length RTKs has enabled the development of two complimentary assay platforms. First, we present a competitive displacement assay that uses a europium (Eu)-labeled anti-GFP antibody and an AlexaFluor® 647-labeled active site probe to characterize compound binding to the RTK in a lysate-based format. Here, the GFP moiety serves as an epitope tag to allow for very specific and sensitive detection of compound binding, discriminating compounds with sub-nanomolar affinity. Using the same BacMam RTK-GFP constructs, we have also developed a simple, addition-only, cell-based method to detect auto-phosphorylation of the RTK via TR-FRET between a terbium (Tb)-labeled anti-phospho-tyrosine antibody and the GFP moiety. Together, these tools can be used in a high-throughput screening format with a fluorescence-based readout to more fully characterize compound efficacy against full-length RTKs expressed in native cellular contexts.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3881. doi:1538-7445.AM2012-3881
Abstract 3887: A screen for new cancer specific drugs that target centrosome clustering
Authors: Kawamura Eiko , Fielding Andrew , Kannan Nagarajan , Eaves Connie , Roberge Michel , Dedhar Shoukat 
Published: August 2012

Abstract
Most normal cells have two centrosomes that form bipolar spindles during mitosis, while cancer cells often contain extra centrosomes. Such cancer cells then achieve bipolar division by clustering centrosomes into two functional poles. Our lab has previously shown that inhibition of Integrin-linked kinase, which is a signaling and scaffold protein in focal adhesion and is also a centrosomal protein, results in scattered centrosomes and multipolar spindles, which leads to cancer specific cell death (Fielding et al., 2011, 30:521-534, Oncogene). A major problem with clinically used anti-mitotic drugs, such as taxol, is their toxicity in normal cells. Our goal is to develop new drugs that act specifically on cancer cells through targeting extra centrosomes without affecting normal cells. To discover new drugs, we established a high-content screen that automatically detects cells with de-clustered centrosomes. BT-549, a breast cancer cell line, was used for the screen, since it is known to effectively cluster centrosomes. The Canadian Chemical Biology Network enabled us to test chemical libraries predicted to have drug-like properties. For screening, cells were grown in 96 well plates, treated with test compounds for 5 hours, and subjected to immunofluorescence to examine centrosome arrangement in mitotic cells. Images were taken and analyzed with an automated fluorescence imager, Cellomics Array Scan VTI. We have thus screened over 6000 compounds from which we identified 18 hits. Fifteen of these were confirmed to inhibit centrosome clustering in the cells cultured under standard conditions, and induce an arrest in mitosis. Three compounds are structurally similar, suggesting a common structural motif as preventing centrosome clustering. To compare the effects of these drugs on normal and cancer cells, the viability of several breast and other cancer cell lines, an immortalized model of normal human mammary epithelial cells (MCF10A), and freshly isolated primary normal human mammary epithelial cells were examined after two days of drug treatment using the MTT assay. These comparisons identified some compounds that selectively reduced the viability of cancer cells, but not normal mammary epithelial cells suggesting their potential for cancer-specific therapy. These small molecules are currently being tested in orthotopic and xenograft models, and will be studied for target identification and mechanism of action for further drug development.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3887. doi:1538-7445.AM2012-3887
Abstract 3894: Discovery, synthesis and SAR studies of a novel nonpeptidic reversible proteosome inhibitor with low nano-molar chymotrypsin like activity
Authors: Ozcan Sevil , Kazi Aslamuzzaman , Sun Nan N. , Daniel Kenyon , Guida Wayne C. , Lawrence Harshani R. , Sebti Said M. 
Published: August 2012

Abstract
The development of proteosome inhibitors for CT-L activity has been the subject of considerable interest in the treatment of cancer due to its critical role in maintaining homeostasis and hence its key position in many cellular processes. Proteosome inhibitors are classified as reversible or irreversible inhibitors according to their chemical structure and their mechanism of inhibition. Irreversible inhibitors possess a chemically reactive group, such as aldehydes, boronic acids, epoxy ketones that react with catalytic Thr1-O covalently, where as reversible inhibitors inhibit proteosome non-covalently via H-bond interactions (electrostatic and/or van der Waals). Bortezomib, a peptide boronic acid analog, is the first clinically approved proteosome inhibitor and is found to be an irreversible or slow reversible inhibitor that forms covalent bond with the N-terminal nucleophilic Thr1 in the β5 subunit of the proteasome. Reversible proteosome inhibitors (peptidic molecules) reported to date inhibit the proteosome non-covalently. Similar to Bortezomib, MLN9708 is a modified dipeptidyl boronic acid, which hydroylses immediately in plasma to MLN2238, is a potent, reversible and specific inhibitor of the chymotrypsin-like subunit of the proteasome. Small molecules as reversible proteosome inhibitors are less extensively investigated. In this study, we present the discovery of PI-184, non-peptidic small molecule with a pyridine and oxadiazole pharmacore as a reversible, potent and a novel proteasome inhibitor. Structure Activity relationship (SAR) guided synthesis of analogs around PI-184 led to the discovery of highly potent compounds with in-vivo chymotrypsin-like inhibitory activity. Here we present the detailed SAR, in-vitro and in-vivo proteasome activity of PI-184 class of compounds as potential anti-cancer agents.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3894. doi:1538-7445.AM2012-3894
Abstract 3896: New heat shock protein 90 inhibitors based on the 1,2,3-triazole structure
Authors: Giannini Giuseppe , Taddei Maurizio , Ferrini Serena , Alloatti Domenico , Brunetti Tiziana , Vincenti Silvia , Vesci Loredana , Castorina Massimo , Milazzo Ferdinando , Cervoni Maria Luisa , Barbarino Marcella , Guglielmi Mario Berardino , Pisano Claudio , Cabri Walter 
Published: August 2012

Abstract
Hsp90 (heat shock protein 90) is a component of a molecular chaperone complex, involved in the folding, maturation and stabilisation of key signalling proteins which control cell proliferation, survival and transformation. It works by modulating a set of cancer-associated proteins, that are often over-expressed and/or mutated in tumor cells, collectively referred as ‘‘clients’’. Inhibition of Hsp90 causes destabilization and eventual degradation of client proteins involved in cell cycle, tumor growth, angiogenesis and apoptosis. Therefore, their modulation results in suppression of tumor growth by multiple parallel mechanisms. Consequently, inhibition of Hsp90 is believed to be a potential target for cancer therapy, also supported by a number of molecules currently under investigation in different phase of clinical trials. We recently synthesized 1,4,5-trisubstituted-1,2,3-triazole derivatives and surprisingly found that these new molecules demonstrated to be extremely active both on the biological target and on human tumor cell lines, even at nanomolar concentration. 3D QSAR analysis was also performed in order to rationalize HSP90 binding data. The overall profile of this new class of 1,2,3-triazoles, including stereoselective synthesis and a comprehensive pharmacological in vitro characterization, will be presented.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3896. doi:1538-7445.AM2012-3896
Abstract 3897: Design and preclinical evaluation of single agents with antitubulin and antiangiogenic activity as antitumor agents
Authors: Gangjee Aleem , Pavana Roheeth Kumar , Zhang Xin , Ihnat Michael , Hamel Ernest 
Published: August 2012

Abstract
Agents that interfere with microtubules are important antitumor agents. Tumor angiogenic mechanisms that are vital for tumor growth and metastasis are targeted by antiangiogenic agents. Antiangiogenic agents are usually not tumoricidal but are mainly cytostatic. Combination chemotherapy with antiangiogenic and cytotoxic agents has shown significant promise, and several studies with such combinations are in progress in the clinic. Single agents with both antiangiogenic activities and cytotoxicity could afford single agents that would circumvent the pharmacokinetic problems of multiple agents, avoid drug-drug interactions, be used at lower doses to alleviate toxicity, be devoid of overlapping toxicities, and delay or prevent tumor cell resistance. We have designed, synthesized and evaluated two novel compounds, AG119 and AG425, that inhibit both vascular endothelial growth factor receptor-2 (VEGFR-2) for antiangiogenic effects and tubulin for cytotoxic effects in single agents. Both compounds have comparable potency as sunitinib and semaxinib against cell lines overexpressing VEGFR-2 and were equipotent with semaxinib on VEGFR-2 mediated endothelial cell proliferation, indicating a functional effect of these agents on VEGFR-2 inhibition (IC50: semaxanib 68.2 ± 4.2 µM, AG119: 60.2 ± 2.4 µM, AG425: 52.3 ± 3.8 µM). The novel compounds are effective and potent inhibitors (comparable to combretastatin A-4) of bovine brain tubulin assembly (IC50: combretastatin A-4 1.2 ± 0.01 µM, AG119: 3.5 µM, AG425: 1.9 ± 0.1 µM). The compounds were potent inhibitors of [3H]colchicine binding to tubulin, indicating that these compounds, particularly AG425, bind at the colchicine site. These agents cause microtubule depolymerization, arrest cells in late G2/M phase and trigger apoptotic cell death. Moreover, AG119 and AG425 are potent anticancer compounds in aggressive basal like breast cancer (BLBC) and triple negative breast cancer (TNBC) cell lines. The novel compounds showed sensitivity in taxol resistant cells, indicating that they are unlikely to be Pgp or MRP substrates [unlike taxol], and these compounds possess relative selectivity of kill on tumor versus normal cell types. In vivo these agents reduce tumor size and vascularity in two flank xenograft models [the BLBC MDA-MB-435 model and U251 glioma model] in immunodeficient mice, superior to docetaxel and sunitinib, without overt toxicity to the animals.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3897. doi:1538-7445.AM2012-3897
Abstract 3898: NOSH-aspirin: A novel nitric oxide- and hydrogen sulfide-releasing hybrid for treatment of colon cancer
Authors: Lee Diana H. , Nesbitt Diandra E. , Chattopadhyay Mitali , Kodela Ravinder , Kashfi Khosrow 
Published: August 2012

Abstract
Introduction: In our search for a better non-steroidal anti-inflammatory drug (NSAID) we have synthesized a series of nitric oxide-, and hydrogen sulfide-releasing hybrid compounds that have IC50s for cell growth inhibition in the low nano-molar range. We have termed these NOSH compounds. Our lead agent, NOSH-aspirin is in the order of 100,000 to 250,000-fold more potent than aspirin in inhibiting colon cancer cell growth over a 24-72h time period. NOSH-aspirin although highly potent, has very limited cyto-toxicity, lactate dehydrogenase (LDH) release at 4 times its IC50 for cell growth inhibition was less than 10% at 24h. Here we report our results on the effects of NOSH-aspirin treatment on established tumors in a xenograft model of human colon cancer cells. Methods: Compound: NOSH-aspirin was synthesized and purified by us with 1H-NMR verification. Cell line: HT-29 human adenocarcimoma; Xenografts: Male athymic SKID mice were implanted s.c. in the right flank with HT-29 (2 x 106) cells suspended in 50% Matrigel, after 10 days the animals were randomly divided into 4 groups (N = 7/group) and gavaged daily with NOSH-aspirin (25 or 50 or 100 mg/kg body weight) or vehicle. Tumor volume and animal weight were recorded every 3 days. After 3 weeks of treatment, mice were sacrificed, tumors excised, weighed, and fixed in 10% buffered formalin for immunohistochemical studies. Results: NOSH-aspirin had no effect on the weight of the mice even at the highest concentration used. There were no overt signs of toxicity. It dose-dependently reduced tumor mass by 50 ± 7%, 75 ± 5%, 90 ± 3 % at 25, 50, and 100 mg/kg, respectively. Tumor volume was also dose-dependently reduced as a function of time. NOSH-aspirin inhibited growth of HT-29 colon cancer cells xenografts as a result of reduced proliferation (decreased PCNA expression), and induction apoptosis (increased number of TUNEL positive cells). NF-κB, activated in control xenografts was significantly inhibited by NOSH-aspirin. Conclusions: NOSH-aspirin has potent anti-cancer affects and merits further evaluation.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3898. doi:1538-7445.AM2012-3898
Abstract 3899: NOSH compounds: Nitric oxide- and hydrogen sulfide-releasing hybrids, a new class of anti-inflammatory pharmaceuticals
Authors: Vannini Federica , Kim Han-Bee , Chattopadhyay Mitali , Kodela Ravinder , Kashfi Khosrow 
Published: August 2012

Abstract
Introduction: The clinical usefulness of non-steroidal anti-inflammatory drugs (NSAIDs) combined with their potentially life-threatening toxicity has prompted intensive efforts to identify safer alternatives, which will at least maintain their pharmacological properties. The search for a “better NSAID” has resulted in at least two modified hybrids, one that releases nitric oxide (NO-NSAIDs), and another that releases hydrogen sulfide (HS-NSAIDs). These agents although much safer and more potent than their traditional counterparts, have IC50s for cell growth inhibition that are in the micro-molar range. Therefore, we postulated that a new hybrid that incorporated the active parts of each compound may be more potent and effective than either one. Our hypothesis has proved to be correct. We have synthesized a number of NSAID based NOSH (nitric oxide-, hydrogen sulfide-releasing) compounds that release both H2S and NO, and have IC50s for cell growth inhibition in the low nano-molar range. Methods: Compounds: Aspirin based NOSH-1, NOSH-2, NOSH-3, and NOSH-4 were synthesized and purified by us with 1H-NMR verification. Human cancer cell lines: colon (HT-29, HCT 15, SW480), breast (MCF-7, MDA-MB-231, SKBR3), pancreas (BxPC-3, MIA PaCa-2), lung (A549), prostate (LNCap), leukemia (Jurkat). Cell growth: MTT. Anti-inflammatory: paw edema induced by intraplantar injection of 100 µL of 1% carrageenan, paw volumes measured up to the tibiotarsal joint immediately prior to carrageenan injection and thereafter at 1hr intervals up to 6hrs. Drugs were administered orally 1 hr before carrageenan; COX enzyme: immunoblotting, PGE2: ELISA Results: All NOSH compounds were more effective than aspirin in inhibiting the growth of colon, breast, pancreas, prostate, lung, and leukemia cancer cell lines. The IC50s at 24h were in the range 48 nM to 6.5 µM depending on the cell line and the compound. The corresponding IC50 for aspirin was &gt;5000 µM in all cell lines. Our lead compound NOSH-1, had a enhanced potency of &gt;100,000-fold. NOSH-1 dose-dependently decreased the carrageenan-induced edema 1h after administration and maintained this anti-inflammatory effect throughout the experiment (up to 6h). PGE2 levels within the inflamed rat paw were also reduced by NOSH-1. Carrageenan induced both COX-1 and COX-2 expression which were dose-dependently inhibited by NOSH-1. Conclusions: NOSH compounds are a new class of anti-inflammatory agents. NOSH-1, our lead compound is very potent in inhibiting a number of different cancer cell lines of different tissue origin suggesting a tissue independent effect.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3899. doi:1538-7445.AM2012-3899