Abstracts C1 - C8

Category C.  Cancer Biology/ Molecular Biology /Developmental Biology

 

C1.  Curcumin-loaded Polymeric Micelles: Preparation and Effects on FLT3-overexpressing Leukemic Cells

Singkome Tima a, d, Siriporn Okonogi b, Chadarat Ampasavate b, Songyot Anuchapreeda c, and Cory Berkland d

aNanoscience and Nanotechnology Program, Graduate School, Chiang Mai University, Chiang Mai 50200, Thailand.
bDepartment of Pharmaceutical Sciences, Faculty of Pharmacy, Chiang Mai University, Chiang Mai 50200, Thailand.
cDepartment of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand.
dDepartment of Pharmaceutical Chemistry, School of Pharmacy, University of Kansas, Kansas 66047, USA.

Curcumin is one of the most popular natural compounds that have been studied for medicinal purposes especially for leukemia treatment. However, the very low water solubility of curcumin is a major problem of its clinical application. This study aims to enhance the solubility of curcumin by entrapment into polymeric micelles and evaluate their physical properties and effects on FLT3-overexpressing EoL-1 and MV4-11 leukemic cell models. Film hydration method was performed for the preparation of curcumin-loaded polymeric micelles (cur-micelles). Ten formulations of cur-micelles at various ratios of curcumin to polymer (1:10 to 1:100) were prepared and evaluated their properties, including mean particle size, PdI, zeta potential, %LC, %EE, morphology, XRD, DSC, and drug releasing. The inhibitory effects of cur-micelles on leukemic cells were also evaluated using MTT assay. The results showed that, Curcumin is fully dispersible after the curcumin-containing polymer film was rehydrated by PBS, pH 7.5. A clear solution of curcumin-loaded polymeric micelles was observed. All formulations demonstrated the particle size with diameter less than 50 nm. The PdI and zeta potential values ranged from 0.220 to 0.322 and -2.50 to -9.99 mV, respectively. %EE and %LC ranged from 77 to 93% and 0.9 to 7.7%, respectively. Additionally, percentage of cumulative release of curcumin from cur-micelles formulation 1:30 and 1:50 at 96 h were 59.2 and 88.5%, respectively. The XRD spectra and DSC curves revealed that curcumin was molecularly incorporated in the micelles. Moreover, cur-micelles demonstrated an excellent cytotoxic activity in both leukemic cells with IC50 value lower than that of free curcumin. Polymeric micelles, performed in this study, can enhance both the water solubility and the cytotoxicity of curcumin on FLT3-overexpressing leukemic cell. These results are necessary for our further investigation of curcumin’s inhibitory mechanisms on FLT3 expression.

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C2.  Detection of a Novel PIAS1 SUMOylation Site in Hepatocytes

Wenqi Cui1 Nadezhda Gelava2, Todd Williams2, and Jeff L. Staudinger1

1Department of Pharmacology and Toxicology, and  2Mass spectrometry & Analytical proteomics Lab, University of Kansas, Lawrence, Kansas 66045, USA

Covalent modification by SUMO is an important regulator of the functional properties of many proteins implicated in human diseases. While there are many examples of individual specific proteins regulated by SUMOylation, there has been no comprehensive survey of the targets of SUMOylation in a human disease. The major reason for this is the lack of a facile and general method for comprehensive identification and quantitating SUMOylated proteins in cells or live animal models. Mapping specific SUMOylated target substrate proteins by mass spectrometry (MS) is challenging for two main reasons: (1) the SUMO modification is dynamic and occurs with extremely low stoichiometry, and (2) the large size of resulting SUMO peptide chains following tryptic digestion renders target proteins refractory to analysis with MS techniques. To resolve those problems, we genetically engineered two adenoviral expression vectors which encode (1) a modified SUMO protein (His)6-SUMO3Q87R, and (2) the E3 SUMO-ligase enzyme-PIAS1. Through the addition of an N-terminal (His)6 moiety and an X-press epitope, and also by adding a unique trypsin cleavage site at the COOH-terminus, we have created a recognizable short five amino acid SUMO remnant (-QQTGG) that can be easily detected using MS-based methods. Coexpression of the PIAS1 protein drives SUMOylation into the nucleus in primary cultures of hepatocytes. Current preliminary data indicate that these tools are valid and will likely be extremely valuable to multiple investigators in the current KU research community who are interested in studying SUMO-related disease links.

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C3.  LC-MS Determination of prostanglandis, leukotrienes, hydroxyeicosatetraenoic acids in rat colon microdialysate during inflammatory bowel disease

Yunan Wang and Craig Lunte

Department of Chemistry, University of Kansas and Ralph N. Adams Institute for Bioanalytical Chemistry, University of Kansas.

Inflammatory bowel disease (IBD) is a widespread gastrointestinal tract disease. It affects 1.4 million people in the US and 2.2 million people in Europe. IBD can be triggered by an abnormal immune system response. The exogenous antigens activate T-cells, releasing cytokines from the intestinal epithelium. These cytokines can significantly increase pro-inflammatory cytokines, such as the TNF-α. Therefore, cyclooxygenase (COX), lipoxygenase (LOX) enzymatic mediating mechanisms are triggered. The pro-inflammatory prostanglandis (PGs) including 6-ketoprostaglandin F, thromboxane B2, 8-isoprostaglandin F, and prostaglandins F2, E2, and D2 can be used as biomarkers of the COX pathway during the inflammatory response in IBD. Meanwhile, biomarkers from LOX pathway include leukotrienes (LTs) such as leukotriene B4, C4, D4 and E4 and hydroxyeicosatetraenoic acids (HETEs) such as 5-HETE and 15-HETE. Our goal was to develop a LC-MS method to monitor the change of these biomarkers in microdialysis sample to study the roles of LOX and COX enzyme activity in the inflammatory response in IBD.

LC separation was obtained by a Phenomenex Kinetex XB-C18 column (100 mm ×2.1 mm i.d., 1.7 µm particle sized) with binary solvent gradient elution. Solvent A consisted of water and 5 mM formic acid. Solvent B consisted of acetonitrile and 5 mM formic acid. A two-step linear gradient was used: 0-18 minutes solvent B increased from 25 to 80%, and then at 80% from 18 to 25 minutes. Tandem mass spectra were acquired on a Thermo ion-trap mass spectrometer coupled with an ESI interface. The microdialysis probe was implanted into the rat colon. Ringer’s solution was perfused to extract these biomarkers and then the dialysate was analyzed by the LC-MS method.

All the 12 analytes were detected with good LOD, from 1-5 ng/L. The regression coefficients (R2) from the calibration curve (1-50 ng/L) were over 0.999 for PGs and 0.98 for LTs and HETEs.

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C4.  Macroglobulin complement related (Mcr) and Thioester protein 3 (Tep3) demonstrate a requirement for thioester-containing proteins in Drosophila development

Sonia Hall, Cyril Cook, and Robert Ward

Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045

Thioester-containing proteins (TEPs) are conserved among vertebrates and invertebrates and include the complement and macroglobulin families of proteins. TEP proteins have been studied for their role in innate immunity and have been found to aid in the phagocytosis of pathogens. Our lab recently characterized one of the six Drosophila TEP genes, Macroglobulin complement related (Mcr), as having an essential developmental role in the establishment and maintenance of the epithelial septate junction, the invertebrate barrier junction that is functionally analogous to the vertebrate tight junction. Interestingly, a recent examination of the vertebrate TEP gene, CD109, also suggested a developmental requirement, indicating that this protein family may have roles in essential developmental processes, in addition to innate immunity. Our lab is investigating if this dual role is specific to Mcr and CD109 or is a conserved feature of additional TEP genes. To examine this question, we conducted a lethal phase analysis and found that four of the six Drosophila TEP genes contribute to one or more developmental processes. Thioester protein 3 (Tep3) encodes a protein with a signal sequence, a potential integrin binding site, and a complete macroglobulin domain. Mutations in Tep3 result in an extended lethal phase with a variety of terminal phenotypes, including persistent salivary glands. Autophagy dependent programmed cell death is required for the destruction of larval salivary glands during metamorphosis and requires the engulfment receptor Draper. The localization of Draper in salivary glands requires Tep3, suggesting a role for Tep3 in developmental autophagy.

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C5.  Microfluidic Lectin Barcode Array for High-Throughput Differential Glycomic Profiling of Ovarian Cancer Biomarker

Yuqin Shang1and Yong Zeng1,2

1Department of Chemistry,  2Bioengineering Graduate Program, University of Kansas, USA

Protein glycosylation holds the key to understanding the molecular mechanisms of cancer and to developing new biomarkers [1]. Despite its biomedical significance, progress in glycomics has considerably lagged behind genomics and proteomics. While MS-based analyses have shed light on the biological and clinical implications of glycans, low-throughput MS methods have had difficulties in correlating glycosylation aberrations with the pathological status. Similar to DNA and protein microarrays, lectin array offers a simple and high-throughput tool that is complementary to MS, which enables the whole tissue-level studies of human plasma glycome [2]. However, lectin-based assays suffer from an intrinsic limitation of the lectin-glycan interactions that are much weaker than the antibody- antigen affinity. Microfluidics offers a unique engineering solution to address this limitation because of its advantages in leveraging assay efficiency, speed, sensitivity, and throughput for genomic and proteomic analyses [3]. Surprisingly, very limited progress has been made to develop novel microfluidics-based glycomics analysis techniques. Here we report on a microfluidic lectin-barcode chip for high-throughput glycan profiling of cancer biomarkers. We have investigated two formats of lectin arrays using a panel of 16 lectins. Various blocking methods have been studied to suppress the interferences caused by non-specific lectin-glycan interactions and the glycans on the detection antibodies, significantly improving the performance of the antibody-overlay-lectin array. Using a biomarker for ovarian cancer, CA125, we demonstrated the feasibility of the microfluidic lectin barcode assay for glycan profiling of disease biomarkers.

REFERENCES
[1] "Alterations in glycosylation as biomarkers for cancer detection." Reis, C.A., Osorio, H., Silva, L., Gomes, C. & David, L. J. Clin Pathol 63, 322 (2010).
[2] "Shotgun glycomics: a microarray strategy for functional glycomics." Song, X. et al.; Nat Methods 8, 85 (2011).
[3] "Disruptive by design: a perspective on engineering in analytical chemistry." Herr, A.E., Anal Chem 85, 7622 (2013).

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C6.  Novel Amyloid-β-binding Alcohol Dehydrogenase Inhibitor Decreases Cancer Cell Growth Rate

Emily A. Carlson1, Valasani Koteswara Rao1, Rebecca T. Marquez2, Liang Xu2, and Shirley ShiDu Yan1

1Department of Pharmacology and Toxicology, and  2Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045, USA

Cancer comprises a vast set of diseases that are difficult to treat. As cancer cell resistance renders existing therapies ineffective for many patients, it is crucial that new treatments be found. Amyloid-β-binding alcohol dehydrogenase (ABAD) plays a protective role in cells undergoing stress. Across disease states, increased ABAD levels can have a profound and varied impact on cellular health, such as beneficial in Parkinson’s disease and harmful in Alzheimer’s disease. Recently, ABAD overexpression was observed in some prostate and bone cancers, consistently correlating with poor patient prognosis. Also, our past studies revealed that ABAD is increased in breast tumor tissue, indicating that ABAD is involved in many different cancers. Thus, we propose that ABAD overexpression promotes cancer cell development, and ABAD inhibition decreases that growth. The main objectives of our study were to determine the effect of increased ABAD expression on cancer cell growth and migration, and to examine the consequence of ABAD inhibition. We show that cancer cells overexpressing ABAD grow faster in cell culture and an in vivo tumor mouse model. We further demonstrate that the increased growth rate observed in ABAD-overexpressing cancer cells is reduced by inhibition of ABAD with a small molecule inhibitor generated in our laboratory. These results suggest that blockade of ABAD may have potential for limiting tumor growth, which could have a significant impact on cancer treatment.

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C7.  The Retinaldehyde Reductase DHRS3 Is Essential for Preventing the Formation of Excess Retinoic Acid during Embryonic Development

Suya Wanga, Sara E. Billingsa, Keely Pierzchalskib, Naomi E. Butler Tjadencd, Xiaoyan Panga, Paul A. Trainorcd, Maureen A. Kaneb, Alexander R. Moisea,1

aDepartment of Pharmacology and Toxicology, School of Pharmacy, University of Kansas, Lawrence, Kansas, 66045, USA;
bDepartment of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland, 21201, USA;
cStowers Institute for Medical Research, Kansas City, Missouri, 64110, USA;
dDepartment of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas, 66160, USA.

The homeostasis of the vitamin A metabolite, all-trans-retinoic acid (ATRA), is maintained by a precise balance between its synthesis and degradation. The precise levels of ATRA need to be carefully maintained to ensure proper embryonic development. In the current studies, we focus on the role of a DHRS3, a ubiquitously expressed short chain dehydrogenase/ reductase (SDR) enzyme which reduces retinaldehyde to retinol and thus prevents formation of excess ATRA. Here, we investigate the physiological role of Dhrs3 by employing a Dhrs3-deficient mouse model. Ours studies reveal that Dhrs3-deficient embryos exhibit a significant reduction in the levels of retinol and retinyl esters and an increase in ATRA, compared to wild-type embryos. Such an accumulation of ATRA in Dhrs3-/- mice leads to metabolic compensation through both upregulation of the expression of the ATRA catabolic enzyme Cyp26a1 and reduction in the expression of genes involved in ATRA synthesis. Despite such compensation, analysis of gene expression reveals that ATRA signaling pathways are altered in Dhrs3-/- mice. As a result of these alterations, Dhrs3-/- mice present with cardiac malformations, cleft palate and defects in skeletal development and are not viable. Some of the Dhrs3-/- mice also exhibit defects in the patterning and axonal outgrowth of the cranial nerves. We also found that by reducing maternal intake of vitamin A, the skeletal defects observed in Dhrs3-/- embryos were ameliorated. The modulation of the phenotype of Dhrs3-/- mice in response to alterations in vitamin A status demonstrates the causal link between the proposed enzymatic activity of DHRS3 and the phenotype of mice lacking this enzyme. These results provide evidence of the critical function of DHRS3 in reducing retinaldehyde to retinol to safeguard against excess formation of ATRA during embryonic development.

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C8.  The Role of Septate Junction Genes in Drosophila Melanogaster Oogenesis

Haifa Alhadyian and Robert Ward

Department of Molecular Biosciences, University of Kansas

Collective cell migrations are crucial for embryonic development during events such as gastrulation and neurulation. They are also important in adult tissues, for example during wound healing. Inappropriate cell migration can result in severe developmental defects and can contribute to metastasis. The mechanisms that control collective cell movements spatially and temporally are poorly understood. It is also unclear how cells remain attached during collective cell movements, although cell adhesion mediated by E-cadherin at adherens junctions is often suggested. In invertebrate epithelia, septate junctions (SJs) reside just basal to the adherens junction, and contain many adhesion molecules. Our preliminary data indicates that SJ genes are required for a number of morphogenetic events during early embryonic development in the fruit fly, Drosophila Melanogaster. To determine if SJ genes play a role in collective cell migrations at other stages of development, we are studying their function during border cell migration in oogenesis. In stage 9-10 egg chambers the two anterior-most follicle cells recruit 4-8 neighboring cells, delaminate as a cluster and migrate between nurse cells until they reach the oocyte. To investigate the role of SJ genes in border cell migration, we are using the UAS-GAL4 system and RNA interference to specifically knock down the expression of SJ genes in just the migrating border cells or in all the follicle cells at later stages of oogenesis. These studies should shed light on the role of SJ genes in collective cell migration during oogenesis, which may be generally applicable for other developmental events.

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