Molecule Kinase Research Articles

MKP-1 attenuates LPS-induced blood-testis barrier dysfunction and inflammatory response through p38 and IκBα pathways.

Sertoli cells create a local tolerogenic microenvironment to maintain testicular immune privilege especially through the formation of a blood-testis barrier (BTB). However, the molecular mechanisms underlying the immune modulation function and BTB dynamics of Sertoli cells remained elusive. MAP phosphatase (MKP)-1 acts as a crucial negative regulator of the inflammatory response. Nevertheless, the role of MKP-1 in regulating Sertoli cells has not been elucidated. In this study, we have for the first time uncovered distinct cellular localization of MKP-1 in the cells at different stages of mouse testis, and the level of MKP-1 expression was significantly up-regulated by LPS-induced acute testis inflammation. In addition, MKP-1 staining was strongly detected in nuclei and peri-nuclear regions of cytoplasm in the Sertoli cells, and it was presented at Sertoli cell tight junctions (TJs) at stages VII-VIII after LPS treatment. Moreover, we demonstrated that MKP-1 was capable of attenuating LPS-induced decrease of occludin by interaction with p38 MAP kinase and IκBα molecules. Taken together, our data highlight that MKP-1 was an important endogenous suppressor of innate immune responses involved in the regulation of BTB barrier dynamic. This study thus might offer novel targets for treating inflammatory diseases in the testis.

Argon Preconditioning Protects Airway Epithelial Cells against Hydrogen Peroxide-Induced Oxidative Stress

Background: Oxidative stress is the predominant pathogenic mechanism of ischaemia-reperfusion (IR) injury. The noble gas argon has been shown to alleviate oxidative stress-related myocardial and cerebral injury. The risk of lung IR injury is increased in some major surgeries, reducing clinical outcome. However, no study has examined the lung-protective efficacy of argon preconditioning. The present study investigated the protective effects of argon preconditioning on airway epithelial cells exposed to hydrogen peroxide (H₂O₂) to induce oxidative stress. Methods: A549 airway epithelial cells were treated with a cytotoxic concentration of H₂O₂ after exposure to standard air or 30 or 50% argon/21% oxygen/5% carbon dioxide/rest nitrogen for 30, 45 or 180 min. Cells were stained with annexin V/propidium iodide, and apoptosis was evaluated by fluorescence-activated cell sorting. Protective signalling pathways activated by argon exposure were identified by Western blot analysis for phosphorylated candidate molecules of the mitogen-activated protein kinase and protein kinase B (Akt) pathways. Results: Preconditioning with 50% argon for 30, 45 and 180 min and 30% argon for 180 min caused significant protection of A549 cells against H₂O₂-induced apoptosis, with increases in cellular viability of 5-47% (p < 0.0001). A small adverse effect was also observed, which presented as a 12-15% increase in cellular necrosis in argon-treated groups. Argon exposure resulted in early activation of c-Jun N-terminal kinase (JNK) and p38, peaking 10- 30 min after the start of preconditioning, and delayed activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, peaking after 60-90 min. Conclusions: Argon preconditioning protects airway epithelial cells from H₂O₂-induced apoptotic cell death. Argon activates the JNK, p38, and ERK1/2 pathways, but not the Akt pathway. The cytoprotective properties of argon suggest possible prophylactic applications in surgery-related IR injury of the lungs.

Disinhibition of the extracellular-signal-regulated kinase restores the amplification of circadian rhythms by lithium in cells from bipolar disorder patients

Bipolar disorder (BD) is characterized by depression, mania, and circadian rhythm abnormalities. Lithium, a treatment for BD stabilizes mood and increases circadian rhythm amplitude. However, in fibroblasts grown from BD patients, lithium has weak effects on rhythm amplitude compared to healthy controls. To understand the mechanism by which lithium differentially affects rhythm amplitude in BD cells, we investigated the extracellular-signal-regulated kinase (ERK) and related signaling molecules linked to BD and circadian rhythms. In fibroblasts from BD patients, controls and mice, we assessed the contribution of the ERK pathway to lithium-induced circadian rhythm amplification. Protein analyses revealed low phospho-ERK1/2 (p-ERK) content in fibroblasts from BD patients vs. controls. Pharmacological inhibition of ERK1/2 by PD98059 attenuated the rhythm amplification effect of lithium, while inhibition of two related kinases, c-Jun N-terminal kinase (JNK), and P38 did not. Knockdown of the transcription factors CREB and EGR-1, downstream effectors of ERK1/2, reduced baseline rhythm amplitude, but did not alter rhythm amplification by lithium. In contrast, ELK-1 knockdown amplified rhythms, an effect that was not increased further by the addition of lithium, suggesting this transcription factor may regulate the effect of lithium on amplitude. Augmentation of ERK1/2 signaling through DUSP6 knockdown sensitized NIH3T3 cells to rhythm amplification by lithium. In BD fibroblasts, DUSP6 knockdown reversed the BD rhythm phenotype, restoring the ability of lithium to increase amplitude in these cells. We conclude that the inability of lithium to regulate circadian rhythms in BD may reflect reduced ERK activity, and signaling through ELK-1.

New Regulatory Roles of Galectin-3 in High-Affinity IgE Receptor Signaling.

Aggregation of the high-affinity receptor for IgE (FcεRI) in mast cells initiates activation events that lead to degranulation and release of inflammatory mediators. To better understand the signaling pathways and genes involved in mast cell activation, we developed a high-throughput mast cell degranulation assay suitable for RNA interference experiments using lentivirus-based short hairpin RNA (shRNA) delivery. We tested 432 shRNAs specific for 144 selected genes for effects on FcεRI-mediated mast cell degranulation and identified 15 potential regulators. In further studies, we focused on galectin-3 (Gal3), identified in this study as a negative regulator of mast cell degranulation. FcεRI-activated cells with Gal3 knockdown exhibited upregulated tyrosine phosphorylation of spleen tyrosine kinase and several other signal transduction molecules and enhanced calcium response. We show that Gal3 promotes internalization of IgE-FcεRI complexes; this may be related to our finding that Gal3 is a positive regulator of FcεRI ubiquitination. Furthermore, we found that Gal3 facilitates mast cell adhesion and motility on fibronectin but negatively regulates antigen-induced chemotaxis. The combined data indicate that Gal3 is involved in both positive and negative regulation of FcεRI-mediated signaling events in mast cells.

Expression of Focal Adhesion Kinase Molecule by Immunohistochemical Localization in Odontogenic Epithelium of Ameloblastoma and Dental Follicle

Expression of Focal Adhesion Kinase Molecule by Immunohistochemical Localization in Odontogenic Epithelium of Ameloblastoma and Dental Follicle

Abstract P5-02-06: Predicting the response of molecular targeting agents in triple-negative breast cancer cell lines by kinase activities

Abstract Introduction Mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)-AKT pathways are two major hyper-activated cascades in triple-negative breast cancer (TNBC) that critically regulate cancer progression by enhancing cell survival, proliferation, metastasis, EMT, cancer stem cell regulate, and transformation. While many therapeutic agents targeting kinases in these pathways are being developed, the development of predictor of response for such agents are critical to successfully translate them into the clinic. Genomic analysis (amplification, deletion of mutation) is one of the prediction methods. However, these technologies do not always reflect the intrinsic functionalities/activities of the kinase molecules. Therefore, we hypothesized that kinase activity predicts the response to the targeted therapy in TNBC. Materials and methods Seventeen TNBC cell lines were used in this study. To analyze cell growth inhibition, cells were incubated for 72 h with various concentrations of trametinib or wortmannin, then processed for sulforhodamine B (SRB) staining assay. To measure MEK or PI3K enzymatic activity, TNBC cell lines were lysed and immunoprecipitated with magnetic beads conjugated with MEK antibody or with PI3K p110α antibody. Kinase reaction buffer including respective substrate and ATP was added to the immunoprecipitates and incubated for 120 minutes at 37 °C. Resultant ADP was quantified by HPLC and determined MEK and PI3K activities. Protein mass of MEK, PI3K, phospho-MEK and phospho-PI3K were determined by Western Blot analysis. Total protein amount was measured by A280. Lactate dehydrogenase (LDH) activity was measured by N-assay L LDH Nittobo. Total protein and LDH were used to normalize MEK and PI3K activities for the further analysis. Results Seventeen TNBC cell lines were classified into 4 groups depending on pattern of inhibition to two inhibitors as follows; Wortmannin (PI3K inhibitor) sensitive group (W, 2/17), Trametinib (MEK inhibitor) sensitive group (T, 2/17), Both sensitive group (S, 5/17) and Resistant group (R, 8/17). We found that ratio of PI3K activity and MEK activity showed good agreement to the cell classification (PPV [Wortmannin]: 67 %, PPV [Trametinib]: 33 %, NPV: = 100 %). The other parameters; enzymatic activity of MEK or PI3K, protein mass of MEK, PI3K, phospho-MEK, or phospho-PI3K, ratios of the protein mass, and the phospho-protein did not show statistically significant agreement to the classification. Mutational status and enzymatic activities or cell classification had no correlation. Additionally, MEK activity correlated to downstream phospho-ERK expression level (R = 0.7309). Conclusion Our results show that relative activity of two relevant kinases in the signaling cascade could predict the cell lines that will not respond to molecular targeting agents against corresponding cascades. Our concept should be warranted in the clinical study with statistically sufficient number of patients. Citation Format: Sato N, Wakabayashi M, Lee J, Lim B, Ueno NT, Ishihara H. Predicting the response of molecular targeting agents in triple-negative breast cancer cell lines by kinase activities. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-02-06.

Fast Closuer of Long Loops at the Initiation of a Protein Folding Pathway

What are the initial steps in the folding of globular proteins? In order to test the hypothesis that long loop closure transitions are part of the initiation of the folding pathways of globular proteins, our groups are studying the initiation of the folding transition of a model protein by time resolved excitation energy transfer (trFRET) detected fast kinetics experiments. Site specific double labeling is used to study the timing of conformational transitions of individual loop forming chain segments at the µs time regime. Previously, it was shown that at least three long loops in the Escherichia coli adenylate kinase (AK) molecule close within the first 5 ms of the 6 seconds folding kinetics of AK. We applied a continuous flow based double kinetics experiment. These measurements enabled us to obtain µs series of transient time dependent distributions of distances between the ends of the labeled loops. Analysis of the trFRET experiments shows that the N terminal loop (loop I) is closed within less than 60 µs after the initiation of refolding. Loop II is also mostly closed within that time step but shows an additional small reduction of the mean end to end distance in a second phase at a rate of 0.005µs−1. This second phase can either reflect tightening of a loosely closed loop in the ensemble of conformers or may reflect two subpopulations in the ensemble, which differ in the rate of closure of loop II, but not in the rate of closure of loop I. This study shows the very fast closure of long loops in the otherwise disordered backbone and fine details of the very early hidden pre-transition state stepsthat are essential for the fast and efficient folding of the protein molecule.

C-glycosyl flavonoid orientin improves chemically induced inflammatory bowel disease in mice

Orientin is a C-glycosyl flavonoid found abundantly in some fruits and herbs. The present study demonstrated that orientin (50 and 100 mg/kg/d) alleviated the severity of dextran sulphate sodium (DSS)-induced colitis in mice by decreasing the activity of myeloperoxidase (MPO) and the production of cytokines. Concomitant up-regulation of toll-like receptor (TLR)-4 after DSS exposure was associated with an increase in the activation of myeloid differentiation factor 88 (MyD88), IL-1 receptor-associated kinase (IRAK)-1, nuclear factor kappa B (NF-kB) p65, as well as the mitogen-activated protein kinase (MAPK) molecules (ERK1/2, JNK and p38), and this upregulation was reversed by orientin administration. Moreover, orientin inhibited the nuclear translocation of NF-kB p65, the activity of NF-kB-luciferase, and the expression of NF-kB target genes. These findings suggest that orientin attenuates experimental inflammatory bowel disease (IBD) via suppression of TLR4 and inactivation of NF-kB and MAPK pathways.

Molecular links between early energy metabolism alterations and Alzheimer's disease.

Recent studies suggest that the neurobiology of Alzheimer's disease (AD) pathology could not be explained solely by an increase in beta-amyloid levels. In fact, success with potential therapeutic drugs that inhibit the generation of beta amyloid has been low. Therefore, due to therapeutic failure in recent years, the scientists are looking for alternative hypotheses to explain the causes of the disease and the cognitive loss. Accordingly, alternative hypothesis propose a link between AD and peripheral metabolic alteration. Then, we review in depth changes related to insulin signalling and energy metabolism in the context of the APPSwe/PS1dE9 (APP/PS1) mice model of AD. We show an integrated view of the changes that occur in the early stages of the amyloidogenic process in the APP/PS1 double transgenic mice model. These early changes affect several key metabolic processes related to glucose uptake and insulin signalling, cellular energy homeostasis, mitochondrial biogenesis and increased Tau phosphorylation by kinase molecules like mTOR and Cdk5.

LEFTY2 Controls Migration of Human Endometrial Cancer Cells via Focal Adhesion Kinase Activity (FAK) and miRNA-200a

Background: LEFTY2, a suppressor of cell proliferation, tumor growth, regulator of stemness and embryonic differentiation, is a negative regulator of cancer cell reprogramming. Malignant transformation may lead to migration requiring loss of adhesion and gain of migratory activity. Signaling involved in the orchestration of migration, proliferation and spreading of cells include focal adhesion kinase (FAK) and adhesion molecule E-cadherin. Aims: The present study explored whether LEFTY2 influences the proliferation marker MKi67, FAK activity, E-cadherin abundance and migration of Ishikawa human endometrial carcinoma cells. Moreover, the study explored the involvement of microRNA-200a (miR-200a), which is known to regulate cellular adhesion by targeting E-Cadherin. Methods: FAK activity was estimated from FAK phosphorylation quantified by Western blotting, migration utilizing a wound healing assay, miR-200a and MKi67 expression levels utilizing qRT-PCR, cell proliferation and apoptosis using BrdU and Annexin V staining, respectively, and E-Cadherin (E-Cad) abundance, using confocal microscopy. Results: LEFTY2 (25 ng/ml, 48 hours) treatment was followed by decrease of MKi67 expression, FAK activity and migration. LEFTY2 upregulated miRNA-200a and E-Cad protein level in Ishikawa cells. The effect of LEFTY2 on migration was mimicked by FAK inhibitor PF 573228 (50 µM). Addition of LEFTY2 in the presence of PF-573228 did not result in a further significant decline of migration. Conclusion: In conclusion, LEFTY2 down-regulates MKi67 expression and FAK activity, up-regulates miR-200a and E-cadherin, and is thus a powerful negative regulator of endometrial cell proliferation and migration.

High-throughput analysis of kinase inhibitor drugs on cardiac function using engineered heart tissue constructs

Event Abstract Back to Event High-throughput analysis of kinase inhibitor drugs on cardiac function using engineered heart tissue constructs Genevieve Conant1, 2, Yimu Zhao2 and Milica Radisic1, 2 1 University of Toronto, Institute of Biomaterials and Biomedical Engineering, Canada 2 University of Toronto, Chemical Engineering and Applied Chemistry, Canada Introduction: A serious concern in health care today is the unintentional long-term effects of pharmaceuticals on the heart, which are not detected during clinical trials. These side effects are burden to health care and can potentially waste millions of dollars in research should the drug be recalled. Tyrosine kinase inhibitors (TKIs) are commonly used as highly specific cancer targeting agents for their ability to prevent tyrosine kinase molecules from activating signaling pathways that regulate cell growth, differentiation, metabolism, migration, and programmed cell death[1]. Due to the highly metabolic nature of cardiac tissue, cardiomyocytes are sensitive to perturbations in mitochondrial function. As such, TKIs have been found to have severe adverse effects on cardiac tissue, however they are poorly understood. Although the human genome has been mapped, and 500 different kinases have been identified, only 50 have been thoroughly studied [2]. High-throughput preclinical screening of over 300 tyrosine kinase-inhibiting molecules could offer much information as to their cardiovascular effects and prevent any further strain on the health care system. We hypothesized that a high-fidelity model for evaluating the physiological effects of TKIs on human myocardium could be established using high-content screening assays and engineered heart tissue constructs, termed BioWires. Methods and Materials: Over 300 TKIs were screened on human cardiomyocytes plated as a monolayer at various concentrations. High-content screening assays from Molecular Devices were used sequentially to assess cell viability and basic cardiac function. TKIs that did not cause detrimental effects to cardiac tissue and cell viability were examined using our 3D BioWire platform. These BioWires are micro-scale cardiac tissues mimicking physiological tissue that consist of human cardiomyocytes seeded onto polymer wires and electrically stimulated in order to promote tissue maturation[3]. Fluorescent image microscopy was used to examine tissue contraction and calcium transient profiles. Particle-tracking software was developed to measure the deflection of the polymer wire, which can be directly related to force of contraction through basic engineering statics. Results and Discussion: Preliminary testing using human embryonic stem cell-derived cardiomyocytes (hES-CM) and human induced pluripotent stem cell-derived cardiomyocytes (iPS-CM) demonstrated that the two assays from Molecular Devices could be used sequentially with no detrimental effect on cardiac function. As well, the effects of known chronotropic and inotropic drugs were examined, and their effect could be quantified on our platform. In the future, TKIs that showed the greatest effect on cardiac function could be further studied to determine their cellular mode of action. Conclusion: In conclusion, this system was capable of providing much needed information regarding the effects of tyrosine kinase inhibiting molecules on the heart, and could also be applied to high-throughput screening of other molecules.

Sequential Closure of Loop Structures Forms the Folding Nucleus during the Refolding Transition of the Escherichia coli Adenylate Kinase Molecule.

The ensemble of conformers of globular protein molecules immediately following transfer from unfolding to folding conditions is assumed to be collapsed though still disordered, as the first steps of the folding pathway are initiated. In order to test the hypothesis that long loop closure transitions are part of the initiation of the folding pathway, our groups are studying the initiation of the folding transition of a model protein by time-resolved excitation energy transfer (trFRET) detected fast kinetics experiments. Site-specific double labeling is used to study the timing of conformational transitions of individual loop forming chain segments at the microsecond time regime. Previously, it was shown that at least three long loops in the Escherichia coli adenylate kinase (AK) molecule close within the first 5 ms of folding of AK, while the main global folding transition occurs in a time regime of seconds. In order to enhance the time resolution of the kinetics experiments to the microsecond time regime and determine the rate of closure of the two N terminal loops (loop I residues 1-26 and loop II residues 29-72), we applied a continuous flow based double kinetics experiment. These measurements enabled us to obtain a microsecond series of transient time dependent distributions of distances between the ends of the labeled loops. Analysis of the trFRET experiments show that the N terminal loop (loop I) is closed within less than 60 μs after the initiation of refolding. Loop II is also mostly closed within that time step but shows an additional small reduction of the mean end-to-end distance in a second phase at a rate of 0.005 μs(-1). This second phase can either reflect tightening of a loosely closed loop in the ensemble of conformers or may reflect two subpopulations in the ensemble, which differ in the rate of closure of loop II, but not in the rate of closure of loop I. This study shows the very fast closure of long loops in the otherwise disordered backbone and fine details of the very early hidden pretransition state steps that are essential for the fast and efficient folding of the protein molecule.

Two FGF Receptor Kinase Molecules Act in Concert to Recruit and Transphosphorylate Phospholipase Cγ

The molecular basis by which receptor tyrosine kinases (RTKs) recruit and phosphorylate Src Homology 2 (SH2) domain-containing substrates has remained elusive. We used X-ray crystallography, NMR spectroscopy, and cell-based assays to demonstrate that recruitment and phosphorylation of Phospholipase Cγ (PLCγ), a prototypical SH2 containing substrate, by FGF receptors (FGFR) entails formation of an allosteric 2:1 FGFR-PLCγ complex. We show that the engagement of pTyr-binding pocket of the cSH2 domain of PLCγ by the phosphorylated tail of an FGFR kinase induces a conformational change at the region past the cSH2 core domain encompassing Tyr-771 and Tyr-783 to facilitate the binding/phosphorylation of these tyrosines by another FGFR kinase in trans. Our data overturn the current paradigm that recruitment and phosphorylation of substrates are carried out by the same RTK monomer in cis and disclose an obligatory role for receptor dimerization in substrate phosphorylation in addition to its canonical role in kinase activation.

MiR-143 suppresses epithelial-mesenchymal transition and inhibits tumor growth of breast cancer through down-regulation of ERK5.

Epithelial-mesenchymal transition (EMT) plays a pivotal role in the development of cancer invasion and metastasis. Many studies have significantly enhanced the knowledge on EMT through the characterization of microRNAs (miRNAs) influencing the signaling pathways and downstream events that define EMT on a molecular level. In this study, we found that miR-143 suppressed EMT. Up-regulating miR-143 enhanced E-cadherin-mediated cell-cell adhesion ability, reduced mesenchymal markers, and decreased cell proliferation, migration, and invasion in vitro. In vivo, the xenograft mouse model also unveiled the suppressive effects of miR-143 on tumor growth. Additionally, we demonstrated that up-regulating extracellular signal regulated kinase 5 (ERK5) was associated with poor prognosis of breast cancer patients. Moreover, we observed an inverse correlation between miR-143 and ERK5 in breast cancer tissues. miR-143 directly targeted seed sequences in the 3'-untranslated regions of ERK5. Furthermore, we revealed that the downstream molecules of glycogen synthase kinase 3 beta (GSK-3β)/Snail signaling were involved in EMT and modulated by ERK5. In summary, our findings demonstrated that miR-143 down-regulated its target ERK5, leading to the suppression of EMT induced by GSK-3β/Snail signaling of breast cancer. © 2015 Wiley Periodicals, Inc.

Disturbed T Cell Signaling and Altered Th17 and Regulatory T Cell Subsets in the Pathogenesis of Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of autoantibodies against nuclear components. Circulating immune complexes of chromatin and autoantibodies deposit in various tissues leading to inflammation and tissue damage. It has been well documented that autoimmunity in SLE depends on autoreactive T cells. In this review, we summarize the literature that addresses the roles of T cell signaling, and Th17 and regulatory T cells (Tregs) in the development of SLE. T cell receptor (TCR) signaling appears to be aberrant in T cells of patients with SLE. In particular, defects in the TCRζ chain, Syk kinase, and calcium signaling molecules have been associated with SLE, which leads to hyperresponsive autoreactive T cells. Furthermore, in patients with SLE increased numbers of autoreactive Th17 cells have been documented, and Th17 cells appear to be responsible for tissue inflammation and damage. In addition, reduced numbers of Tregs as well as Tregs with an impaired regulatory function have been associated with SLE. The altered balance between the number of Tregs and Th17 cells in SLE may result from changes in the cytokine milieu that favors the development of Th17 cells over Tregs.

Binding site identification and role of permanent water molecule of PIM-3 kinase: A molecular dynamics study

The kinome is a protein kinase complement of the human genome, categorized as serine/threonine and tyrosine kinases. These kinases catalyze phosphorylation reaction by using ATP as phosphoryl donor. Proviral Integration Site for Moloney Murine Leukemia Virus (PIM) kinase encodes serine/threonine protein kinases that recognized as proto-oncogene, responsible for rapid growth of cancerous cells. It is implicated in cell survival and function via cell cycle progression and its metabolism. PIM-3, sub-member of PIM kinases is a proto-oncogene, its overexpression inhibits apoptosis, and results in progression of hepatocellular carcinoma. PIM-3 is considered as a promising drug target but attempts to develop its specific inhibitors is slowed down due to the lack of 3D structure by any experimental technique. In silico techniques generally facilitate scientist to explore hidden structural features in order to improve drug discovery. In the present study, homology modeling, molecular docking and MD simulation techniques were utilized to explore the structure and dynamics of PIM-3 kinase. Induction of water molecules during molecular docking simulation explored differences in the hinge region between PIM-1 and PIM-3 kinases that may be responsible for specificity. Furthermore, role of water molecules in the active site was also explored via radial distribution function (RDF) after a 10ns molecular dynamics (MD) simulations. Generated RDF plots exhibited the importance of water for inhibitor binding through their bridging capability that links the ligand with binding site residues.

The anti-inflammatory effect and potential mechanism of cardamonin in DSS-induced colitis.

Cardamonin is a naturally occurring chalcone with strong anti-inflammatory activity. However, the direct effect of cardamonin on intestinal inflammation remains elusive. In the present study, we found that cardamonin markedly ameliorated dextran sulfate sodium-induced mouse body weight loss, diarrhea, colon shortening, spleen swelling, and histological damage, which correlated with a decline in the activity of myeloperoxidase and the production of nitric oxide, tumor necrosis factor-α and interleukin-6 in the colon. The upregulation of toll-like receptor 4 after dextran sulfate sodium treatment was associated with an increase in the activation of myeloid differentiation factor 88, interleukin-1 receptor-associated kinase-1, nuclear factor-κB (NF-κB) p65, inhibitor κBα, and inhibitor κB kinase-α/β, as well as the mitogen-activated protein kinase molecules of extracellular signal-regulated kinase and c-Jun NH2-terminal kinase, and this upregulation was reversed by cardamonin administration. Moreover, cardamonin blocked the nuclear translocation of NF-κB p65, inhibited NF-κB-luciferase activity, and downregulated NF-κB target genes expression. The present study clearly demonstrates a beneficial effect of cardamonin on experimental inflammatory bowel disease via a mechanism associated with suppression of toll-like receptor 4 expression and inactivation of NF-κB and mitogen-activated protein kinase pathways. This study may give insight into the further evaluation of the therapeutic potential of cardamonin or its derivatives for human inflammatory bowel disease.

Abstract 3540: Novel targeted combinational therapies for melanoma

Abstract The BRAFV600E mutation is frequently observed in over 60% of melanomas. Development of the small molecule inhibitor PLX4032, which specifically targets the mutated BRAF protein, offers a novel therapeutic approach by effectively blocking the activation of key signaling molecules of the mitogen-activated protein kinase (MAPK) pathway, such as Mek and Erk, effectively leading to arrest in cell proliferation. Provided that MAPK signaling targets the micropthalmia transcription factor (MITF), which is linked to the expression of various melanoma associated antigens (MAAs), we hypothesized that the inhibition of BRAFV600E can modulate the expression of MAAs. Since MAAs are essential in the development of a melanoma vaccine, due to their increased immunogenicity, altering their expression can be a useful immunotherapeutic approach to targeting melanoma. Using patient derived melanoma cell lines, which express various MAAs; our laboratory has developed a vaccinia virus based immunotherapeutic vaccine. These melanoma cell lines have been characterized by their status of BRAFV600E mutation and found to be either homozygous or heterozygous for the mutation. Inhibition of two key MAPK pathway molecules, namely Mek and Erk, denotes that the melanoma cell lines are responsive to PLX4032. Phenotypically, the cells’ invasive and migratory properties were also modulated upon treatment with PLX4032. Moreover, we observed that the expression of various MAAs was enhanced upon treatment with PLX4032. An amplification of gp100, MART-1 and tyrosinase expression on the RNA level as well as on the protein level was observed. Lastly, expression of MMAs was conserved in vivo following the analysis of melanoma cell xenografts in BALB/c nude mice. Based on our observations in that PLX4032 modulates MMA expression, further studies are warranted to determine the clinical effectiveness of combinational immunologic therapy using BRAFV600E inhibitors with our vaccinia virus melanoma cell line derived preparation. Citation Format: Robert Suriano, Neha Y. Tuli, Jan Geliebter, Raj K. Tiwari, Marc Wallack. Novel targeted combinational therapies for melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3540. doi:10.1158/1538-7445.AM2015-3540

Polyclonal neural cell adhesion molecule antibody prolongs the effective duration time of botulinum toxin in decreasing muscle strength.

This study aimed to investigate if the effective duration time of botulinum toxin A (Btx-A) could be prolonged by polyclonal neural cell adhesion molecule antibody (P-NCAM-Ab). 175 male SD rats were randomly divided into three major groups: control group (n=25), Btx-A group (n=25), and P-NCAM-Ab groups. P-NCAM-Ab groups were composed of five sub-groups, with 25 rats each in the dose-response study. Muscle strength of rat lower limbs was determined using a survey system. The expressions of muscle-specific receptor tyrosine kinase (MuSK) and neural cell adhesion molecule (NCAM) were determined by real-time polymerase chain reactions (RT-PCR) and western blotting (WB). The muscle strength was significantly decreased by Btx-A in Btx-A/P-NCAM-Ab groups compared with normal control group. Besides, the muscle strength of P-NCAM-Ab group was significantly decreased compared with the Btx-A group. The recovery time of muscle strength in P-NCAM-Ab group was significantly longer compared with Btx-A group. RT-PCR and WB assay showed that PNCAM-Ab delayed the increase of MuSK and NCAM after Btx-A injection. P-NCAM-Ab prolongs the effective duration time of Btx-A in decreasing muscle strength, which could provide a novel enhancement in clinical application.

Targeting protein kinase and DNA molecules by diimine–phthalate complexes in antiproliferative activity

The serendipitous discovery of the anticancer drug cisplatin cemented medicinal inorganic chemistry as an independent discipline in 1960s. DNA and protein kinases are one of the major intracellular targets of many anticancer drugs. It is thus highly desirable to develop metal complexes, either by interacting with DNA or to target alternative cellular machinery such as protein kinases to provide a more effective means of monitoring disease progression. In this study we report the synthesis and characterization of few novel Cu(II) and Zn(II) Knoevenagel condensed metallointercalators incorporating phthalic acid. The intercalation behavior of the complexes with DNA is confirmed by spectral and analytical experiments. Due to the promising performance of DNA interaction efficacy of Cu(II) complexes 1–4, their in vitro and in vivo anticancer properties are explored on various cancerous cell lines which reveal that they exhibit substantial anticancer activity without affecting the normal cells. It is found that the complex 1 induces apoptosis in Hep G2 cells. Theoretically, DFT is used to optimize the Cu(II) complexes 1–4 to explore their quantum mechanical properties and to carry out affinity studies against cyclin dependant kinase 2 (CDK2) to understand atomic level interactions. Further, the complex–receptor stability is confirmed by molecular dynamics.