Natural Sciences and Mathematics | Faculty Research Posters

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  Effect of Cyclooxygenase-2 Glycosylation on Downstream Expression of E-Cadherin and β-Catenin in MCF-7 Breast Cancer Cells

  Genna Roan, Grace Alexander, Klarisse Cruz, Janelle Nguyen, and Mary B. Sevigny

  Cyclooxygenase-2 (COX-2) is an enzyme that helps catalyze the formation of prostaglandins, which promote inflammation, pain, and fever and maintain other normal physiological functions throughout the body. However, the overexpression of COX-2 has been found to play a role in various diseases including breast cancer. COX-2 exists as two major glycoforms— 72 kDa and 74 kDa— due to the glycosylation site Asn580 which is glycosylated 50% of the time. Past studies from our lab have shown that this glycosylation regulates COX-2 protein turnover in the cell3. The proteins E-cadherin— a tumor suppressor— and β-catenin— a tumor driver— can be regulated by COX-2 activity

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  Stem Cell Derived Osteoprogenitors and Their Role in Bone Repair Using Morphogenic Activators

  George Washington Brownridge III, Sylvanna Islas, Angelina Miller, and Warren Hoeffler

  Bone constantly cycles through a dynamic process of breakdown and remodeling. Osteoblasts are the specialized mesenchymal stem cells that have a major role in bone formation and the remodeling process whereas their counterpart osteoclasts, handle bone resorption. Embryonic stem cells can be partially differentiated into Progenitor cells, and we worked with #18, a candidate for being an osteoprogenitor that has the potential to respond to morphogenic activators. In the case of bone remodeling, TGF-β 2, BMP-2 and an abundance of CA++ have been shown to be potential activators of differentiation into osteoblasts. Eight different trials were conducted with the cells using different combinations of the three morphogenic activators. After inducing the cells with the activators, we performed Immunohistochemistry (IHC) to analyze the expression of osteocalcin, which is the enzyme that binds calcium to mineralize bone. The cells with varying activator combinations showed different physiology with a variance in the cell shape, structure, and spacing. The greatest results were from the combination of TGF-β 2 and BMP-2, which is consistent with #18 operating as an osteoprogenitor. A 3D construct model of #18 seemed to have a similar structure to that of an osteon, possibly indicating the formation of bone. We took slices of the model and performed an IHC staining for Osteocalcin, Prolyl Hydroxylase (5B5), and Collagen I. We saw a strong positive signal for Col I and 5B5, and a slight positive signal for Osteocalcin. This information confirmed that #18 is an osteoprogenitor and is able to assemble bone.

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  Cytotoxicity Studies of Fijianolide (a.k.a. Laulimalide) Acetylated Derivatives Against Pancreatic Cancer Cell lines to Investigate New Paths For Therapeutic Development

  Tyler A. Johnson, David Coppage, Nicole L. McIntosh, Colon V. Cook, Frederick A. Valeriote, and Phillip Crews

  Opioid receptors belong to the large superfamily of seven transmembrane-spanning (7TM) G protein-coupled receptors (GPCRs). As a class, GPCRs are of fundamental physiological importance mediating the actions of the majority of known neurotransmitters and hormones. The Mu (µ), Delta (δ) and Kappa (MOR, DOR, KOR) opioid receptors are particularly intriguing members of this receptor family as they are the targets involved in many neurobiological diseases such as addiction, pain, stress, anxiety, and depression. To date few marine natural products have been investigated for their neurobiological activities.1 One noteworthy example involves ziconotide (1) from the cone snail Conus magnus.2 Compound 1 was the first marine natural product approved by the FDA and is used for the treatment of pain, marketed under the trade name Prialt® (2004).3 More recently Hamman reported that aaptamine (2) is the first marine natural product to show in vivo anti-depressant activity, however no mechanism of action was proposed.1,4 During a separate collaborative screening project we profiled 96 sponge-derived extracts and discovered that demethyl–aaptamine (3) and demethyl (oxy)–aaptamine (4) were selective DOR agonists using an LC-MS based library of an active methanolic extract coll. no. 92553 FM as shown in Fig. 1. We speculated that the in vivo activity for 2 could thus be linked to the DOR target and to test this hypothesis we conducted the following experiments below.

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  Scale Up Isolation of Aaptamine for In Vivo Evaluation Indicates Its Neurobiological Activity is Linked to the Delta Opioid Receptor

  Nicole L. McIntosh, Eptisam Lambu, Laura Millan-Lobo, Fei Li, Li He, Phillip Crews, Jennifer L. Whistler, and Tyler Johnson

  Opioid receptors belong to the large superfamily of seven transmembrane-spanning (7TM) G protein-coupled receptors (GPCRs). As a class, GPCRs are of fundamental physiological importance mediating the actions of the majority of known neurotransmitters and hormones. The Mu, Delta, and Kappa (MOP, DOP, KOP) opioid receptors are particularly intriguing members of this receptor family as they are the targets involved in many neurobiological diseases such as addiction, pain, stress, anxiety, and depression. Recently we discovered that the aaptamine class of marine sponge derived natural products exhibit selective agonist activity in vitro for the DOP versus MOP receptor. Our findings may explain reports by others that aaptamine demonstrates in vivo anti-depressant effects in mouse models using the Porsolt Forced Swim Test. This project involved the extraction of the sponge Aaptos aaptos (a source of 1), establishing a scale up purification procedure to provide sufficient amounts of 1 (30 mg) for a follow up in vivo evaluation and ultimately confirmation of the structure of 1 using LC-MS and ¹H NMR. The results our purification scheme, chemical analysis and in vivo evaluation of 1 using the Marble burying test in rodents are reported here in and suggest that the in vivo anti-depressant effects of 1 are linked directly to its agonist effects on the DOP receptor.

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  Glycosylation of cyclooxygenase-2 (COX-2) influences the migratory and invasive potential of cells

  Fahad Algaly, Julia Hand, and Mary B. Sevigny

  Prostaglandins are bioactive lipids involved in many physiological functions such as maintenance of the cardiovascular, immune, renal, and central nervous systems. They also play a role in certain diseases like arthritis, cancer, and Alzheimer’s. Cyclooxygenase-2 (COX-2) is the enzyme that catalyzes the initial rate-limiting step in the pathway that converts arachidonic acid to prostaglandins. COX-2 exists as two glycoforms with the molecular weights of 72 and 74 kDa, the latter resulting from the addition of a high mannose chain to the Asn580 residue ~50% of the time. The over-expression of COX-2 is believed to be linked to cancer progression and specifically appears to promote the metastatic phenotype. The objective of this study is to determine the effect of the variable glycosylation of COX-2 at Asn580 on the migratory and invasive potential of cells. COS-1 cells and the breast cancer cell line MCF7 were first transfected with either the wild type or Asn580-mutant human COX-2 gene. Boyden chambers were used to determine the ability of transfected cells to migrate through the membrane, approximately 5x104 cells were plated onto the chambers, and cells were incubated for 16-18 h. Cells were then fixed, stained, visualized and counted. In a previous study, our lab showed that COS-1 cells transfected with the Asn580-mutant COX-2 gene migrated faster through the membrane. In this current study, COS-1 cells transfected with the Asn580-mutant COX-2 gene also had a greater invasive potential; however, MCF7 cells transfected with the wild-type human COX-2 gene migrated faster and also had a greater tendency to invade. The results indicate that the ability of this additional or the lack of glycosylation of COX-2 at Asn580 to either enhance or inhibit the migratory and invasive potential of cells depends greatly on cell type. To confirm this, future studies will be carried out to determine the effect of COX-2 glycosylation on the invasive and the migratory potential of PC-3 and T-47D cancer cell lines.

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  Glycosylation of cyclooxygenase-2 (COX-2) influences the migratory potential of COS-1 cells

  Julia Hand, Renee Dominguez, Miguel Regidor, and Mary B. Sevigny

  A cancer cell’s most threatening property is its ability to metastasize or detach from the primary tumor and migrate to other locations in the body. Previous studies have shown that overexpression of the enzyme cyclooxygenase-2 (COX-2) can increase the metastatic potential of several cell types. COX-2 is the rate-limiting enzyme in the prostanoid biosynthesis pathway, converting arachidonic acid to prostaglandin H2, an important signaling molecule in the body. Glycosylation of COX-2 at the amino acid site Asn580 occurs about 50% of the time, and this results in two forms of the enzyme with molecular weights 72 and 74kDa. The purpose of this study was to investigate the impact of glycosylation of COX-2 at the Asn580 site on the metastatic potential of cells. COS-1 cells were first transfected with either an Asn580-mutant human COX-2 gene or the wild-type human COX-2 gene. A cell migration assay was then carried out on these two groups of cells. Briefly, 5x104 cells were plated onto the membrane of a Boyden Chamber, and cells were incubated for 12 hours. Cells that migrated to the underside of the membrane were fixed, stained, visualized via light microscopy, and counted. Our results revealed that cells transfected with the Asn580-mutant gene migrated faster through the membrane. This indicates that a lack of glycosylation at the Asn580 site of the COX-2 enzyme may lead to an enhanced metastatic potential in cells. Future studies will analyze the effect of variable COX-2 glycoform expression on the migratory potential of tumor cell lines such as MCF-7 and T-47D.