Cell and Developmental Biology Commons^™

Proteasome Inhibition As A Potential Anti-Breast Cancer Therapy: Mechanisms Of Action And Resistance-Reversing Strategies, Rahul Rajesinh Deshmukh

Wayne State University Dissertations

AMPK activation and Ubiquitin Proteasome System (UPS) inhibition have gained great attention as therapeutic strategies for the treatment of certain types of cancers. While AMPK serves as a master regulator of cellular metabolism, UPS regulates protein homeostasis. Although the crosstalk between them is suggested, the relationship between these two important pathways is not very clear. We observed that proteasome inhibition leads to AMPK activation in human breast cancer cells. We report that a variety of proteasome inhibitors activate AMPK in all of the tested cancer cell lines. Our data using Liver Kinase B1 (LKB1)-deficient cancer cells suggests that proteasome inhibitor-induced …

Developmental Evolution Of The Visual System In The Cave-Adapted Small Carrion Beetle Ptomaphagus Hirtus, Jasmina Kulacic

Wayne State University Theses

Extensive research has been done to date on Drosophila and Tribolium eye development; however, not much is known about the molecular basis of development of extremely reduced and modified eyes in cave insects. Ptomaphagus hirtus represents an emerging model system for studying the changes at the molecular level that occurred during the evolutionary adaptation of the eye to the cave environment. Therefore, I have started exploring the morphology and organization of the reduced P. hirtus eyelet, using immunohistochemistry, laser scanning microscopy, and ultrastructural imaging approaches. My findings demonstrate that the adult eyelet lacks ommatidial subdivision and has a thick, clear …

Hrd1 Partners In Endoplasmic Reticulum-Associated Degradation, Aaron Alexander Burr

Wayne State University Dissertations

Protein Quality Control (PQC) comprises cellular pathways that regulate the turnover of short-lived, misfolded proteins. A main component of PQC is Endoplasmic Reticulum (ER)-Associated Degradation (ERAD), which controls the degradation of proteins synthesized in the ER. Aberrations in ERAD have been linked to malignancies such as sarcomas, breast, and pancreatic carcinomas, as well as neurodegenerative disease. The machinery in this system is complex and while significant progress has been made to understand ERAD, it is not clear how the different components come together, or how they are regulated. HRD1 is a resident ubiquitin ligase that has been proposed as a …

Soy Isoflavones Mediate Radioprotection Of Normal Lung Tissue By Modulating The Radiation-Induced Inflammatory Response, Lisa Marie Abernathy

Wayne State University Dissertations

Radiation-induced lung injury (RILI) is caused by an early inflammatory process triggered by damage to lung parenchyma, epithelial cells, vascular endothelial cells and stroma. Initially, oxidative injuries after radiation induce altered expression of pro-inflammatory cytokines. Infiltrating inflammatory cells are stimulated and activated, producing additional mediators, resulting in a cytokine cascade. The expansion and perpetual activation of inflammatory cells, as well as lung parenchyma, lead to clinical pneumonitis. Activated cells produce molecular mediators and growth factors that affect the proliferation and gene expression of lung fibroblasts. This process leads to increased collagen synthesis and deposition, eventually leading to the development of …

Combating Resistance To Epidermal Growth Factor Recpetor Inhibitors In Triple Negative Breast Cancer, Julie Marie Madden

Wayne State University Dissertations

Triple negative breast cancer (TNBC) patients suffer from a highly malignant and aggressive cancer that lacks an effective targeted therapeutic. Although many TNBCs, both in vitro and in vivo, have increased expression of epidermal growth factor receptor (EGFR), EGFR targeted inhibitors, such as gefitinib (GEF), have yet to demonstrate efficacy. Using mass spectrometry to identify pathways that remain activated in the presence of GEF, we found that components of the mTOR signaling pathway remain phosphorylated. While inhibiting mTOR with temsirolimus (TEM) decreased mTOR signaling, EGFR signaling pathways remained activated and the TNBC cell lines continued to proliferate. However, dual treatment …

A Novel Function For 12-Lipoxygenase In C-Met And Integrin Β4 Axis Crosstalk, Elizabeth Tovar

Wayne State University Dissertations

Cancer cell metastasis is the single most threatening occurrence of tumor progression and predicts patient prognosis as well as survival. Invasion can be regulated by the Met receptor tyrosine kinase (c-Met), integrin beta4, and the lipid enzyme, 12-Lipoxygenase (12-LOX). Therefore we sought to determine if beta4, c-MET and 12-LOX comprise a signaling axis. c-Met is implicated in cancer cell dissemination through regulation of invasion in EMT where cell-cell junctions are disturbed to allow motility. Furthermore, beta4 promotes cellular adhesion to the extracellular matrix through hemidesmosomes. However, the homeostatic signaling functions of beta4's cytoplasmic tail can be hijacked by growth factor …

Effect Of Long Term Rapamycin Treatment On Mtor Signalling Network In Colon And Liver Of C57bl/6 Mice, John Sorge

Wayne State University Theses

Many studies have investigated the effects of rapamycin on aging and cancer. However, the effects of long-term rapamycin supplementation on a cancer model have not been performed. This is the first study that investigates the effects of long-term supplementation of rapamycin in a cancer model. ACF analysis of colon tissues in mice showed no significant difference between controls and those supplemented with rapamycin. Factors such as energy balance, cellular environment, PI3K/Akt/mTOR pathway, and more have been assessed in this study. The duration of rapamycin supplementation seems to play an important role in the protection against cancer. Ultimately, this study suggests …

Targeting Cxcr4 With Ctce-9908 Inhibits Prostate Tumor Metastasis, Donald Wong, Pridvi Kandagatla, Walter Korz, Sreenivasa R. Chinni

Wayne State University Associated BioMed Central Scholarship

Abstract

Background

CXCL12/CXCR4 transactivation of epidermal growth factor family receptors in lipid raft membrane microdomains on cell surface is thought to mediate tumor growth and subsequent development of metastatic disease. CTCE-9908 is a known inhibitor of CXCR4. Herein, we tested the efficacy of CTCE-9908 in inhibiting prostate cancer cell growth, invasion, and metastasis.

Methods

We used a panel ofin vitroassays utilizing human prostate cancer cell lines and anin vivoorthotopic prostate cancer model to assess the anti-tumoral activity of CTCE-9908.

Results

We demonstrated that (a) CTCE-9908 treatment resulted in no significant change in the growth of PC-3 and C4-2B cells; (b) …

Recombinant Interleukin-21 Plus Sorafenib For Metastatic Renal Cell Carcinoma: A Phase 1/2 Study, Shailender Bhatia, Brendan, Marc S. Ernstoff, Michael, Elisabeth I. Heath, Wilson H. Miller Jr, Igor Puzanov, David I. Quinn, Thomas, Peter Vanveldhuizen, Kelly, Jeremy, Rachel, Naomi, Sonia, John A. Thompson

Wayne State University Associated BioMed Central Scholarship

Abstract

Background

Despite the positive impact of targeted therapies on metastatic renal cell carcinoma (mRCC), durable responses are infrequent and an unmet need exists for novel therapies with distinct mechanisms of action. We investigated the combination of recombinant Interleukin 21 (IL-21), a cytokine with unique immunostimulatory properties, plus sorafenib, a VEGFR tyrosine kinase inhibitor.

Methods

In this phase 1/2 study, 52 mRCC patients received outpatient treatment with oral sorafenib 400 mg twice daily plus intravenous IL-21 (10–50 mcg/kg) on days 1–5 and 15–19 of each 7-week treatment course. The safety, antitumor activity, pharmacokinetic and pharmacodynamic effects of the combination were …

Regulation Of Nkcc2 Trafficking By Vesicle Fusion Proteins Vamp2 And Vamp3 In The Thick Ascending Limb, Paulo Sebastian Caceres Puzzella

Wayne State University Dissertations

The thick ascending limb (TAL) in the kidney regulates extracellular fluid volume and blood pressure. The Na/K/2Cl cotransporter NKCC2 plays a central role in NaCl absorption by the TAL and blood pressure. NKCC2 trafficking to the apical membrane is a major mechanism to control NKCC2 activity. However, little is known about the proteins that mediate NKCC2 trafficking. Inhibition of the vesicle fusion proteins VAMP2 and VAMP3 blunts the increase in surface NKCC2 expression and NaCl absorption in response to stimulation by cAMP. In other cells, VAMPs mediate fusion of exocytic vesicles with the plasma membrane. Whether VAMP2 and VAMP3 mediate …

Effects Of Altering The Peroxisomal Redox State In Models Of Degenerative Disease, Courtney Rose Giordano

Wayne State University Dissertations

Peroxisomes are important regulators of cellular redox balance and function as a signaling platform to regulate anti-aging metabolic and communication networks. In addition the organelle has emerged as a major player in maintaining cellular ROS at an optimal level. At such levels, these ROS are involved in initiation of signaling cascades and that produce an array of anti-aging and disease processes. However, as cells age over time, ROS amass within the peroxisome and elsewhere in the cell. This leads to an imbalance in oxidative homeostasis and results in compromised signaling networks. The goal of this dissertation was to treat disease …

Mitochondrial Dynamics: Exploring A Novel Target Against Myocardial Ischemia-Reperfusion Injury, Yi Dong

Wayne State University Dissertations

Mitochondrial fusion and fission, collectively termed mitochondrial dynamics, are among the core mechanisms responsible for maintaining mitochondrial health and functional integrity. Dynamin-related protein 1 (DRP1) is a key regulator of mitochondrial fission. Recent studies suggest that i) mitochondrial dynamics, particularly, mitochondrial fission, serves as a mediator of cell fate in the setting of ischemia-reperfusion (IR) injury, and, ii) inhibition of DRP1 and mitochondrial fission provides cardioprotection against IR injury. However, the precise role of DRP1 translocation to mitochondria in the pathogenesis of myocardial ischemia-reperfusion injury has not been established.

Using an established model of hypoxia-reoxygenation (HR) in cultured HL-1 cardiomyocytes, …

Cxcr2 Macromolecular Complex In Pancreatic Cancer: A Potential Therapeutic Target In Tumor Growth, Shuo Wang

Wayne State University Dissertations

The signaling mediated by the chemokine receptor CXC chemokine receptor 2 (CXCR2) plays an important role in promoting the progression of many cancers, including pancreatic cancer, one of the most lethal human malignancies. CXCR2 possesses a consensus PSD-95/DlgA/ZO-1 (PDZ) motif at its carboxyl termini, which might interact with potential PDZ scaffold/adaptor proteins. We have previously reported that CXCR2 PDZ motif-mediated protein interaction is an important regulator for neutrophil functions. Here, using a series of biochemical assays, we demonstrate that CXCR2 is physically coupled to its downstream effector phospholipase C-β3 (PLC-β3) that is mediated by PDZ scaffold protein Na(+)/H(+) exchange regulatory …

Characterization Of Adult Zebrafish Retinal Regeneration Following Two Different Damage Models, Jennifer Lee Thomas

Wayne State University Dissertations

Unlike mammals, zebrafish can regenerate all of their retinal neurons through Müller glial cells, which respond to retinal damage by re-entering the cell cycle to create clusters of progenitor cells. The progenitors continue to proliferate as they migrate to the site of damage, where they ultimately differentiate into new retinal neurons. In contrast, Müller glia of the mammalian retina respond to injury with reactive gliosis, which if persistent, can lead to loss of Müller cell function and devastating vision loss. Despite this, multiple lines of evidence suggest that mammalian Müller glial cells possess a latent ability to regenerate retinal neurons. …

Regulation Of Inositol Biosynthesis And Cellular Consequences Of Inositol Depletion: Implications For The Mechanism Of Action Of Valproate, Rania M. Deranieh

Wayne State University Dissertations

Inositol is a six-carbon cyclitol that is ubiquitous in biological systems. It is a precursor for the synthesis of numerous biologically important compounds, including inositol phosphates and phosphoinositides that are essential for cell function and viability. Inositol compounds play a role in membrane formation, gene regulation, signaling, regulation of ion channels, and membrane trafficking. Furthermore, inositol regulates hundreds of genes, including those involved in the biosynthesis of inositol and phospholipids. While transcriptional regulation of inositol biosynthesis has been extensively studied and well characterized, regulation of inositol biosynthesis at the enzymatic level has not been addressed. The current study shows that …

Junctional Sarcoplasmic Reticulum Protein Processing And Trafficking In Cardiac Tissue And Primary Cultured Cardiomyocytes, Naama Sleiman

Wayne State University Dissertations

Junctional SR is an important and unique ER subdomain in the adult myocyte that releases Ca2+ through the actions of an exclusive set of resident proteins. Cardiac calsequestrin (CSQ2) undergoes two co-translational modifications: N-linked glycosylation on 316Asn, and phosphorylation by protein kinase CK2 on a cluster of 3 serines in its tail. In the heart, CSQ2 molecules undergo extensive mannose trimming by ER mannosidase(s), a posttranslational process that often regulates protein breakdown. To investigate CSQ2 protein processing in cardiomyopathy models, studies were performed to test whether CSQ2 glycan structures would be altered in heart tissue from mongrel dogs induced into …

Resistance Of Human Cytomegalovirus To Cyclopropavir Maps To A Base Pair Deletion In The Open Reading Frame Of Ul97, Brian G. Gentry, Laura E. Vollmer, Ellie D. Hall, Katherine Z. Borysko, Jiri Zemlicka, Jeremy P. Kamil, John C. Drach

Oncology Faculty Publications

Human cytomegalovirus (HCMV) is a widespread pathogen in the human population, affecting many immunologically immature and immunocompromised patients, and can result in severe complications, such as interstitial pneumonia and mental retardation. Current chemotherapies for the treatment of HCMV infections include ganciclovir (GCV), foscarnet, and cidofovir. However, the high incidences of adverse effects (neutropenia and nephrotoxicity) limit the use of these drugs. Cyclopropavir (CPV), a guanosine nucleoside analog, is 10-fold more active against HCMV than GCV (50% effective concentrations [EC₅₀s] = 0.46 and 4.1 μM, respectively). We hypothesize that the mechanism of action of CPV is similar to that …

Hypothesis Driven Single Nucleotide Polymorphism Search (Hydn-Snp-S), Rebecca J. Swett, Angela Elias, Jeffrey A. Miller, Gregory E. Dyson, G. AndréS Cisneros

Chemistry Faculty Research Publications

The advent of complete-genome genotyping across phenotype cohorts has provided a rich source of information for bioinformaticians. However the search for SNPs from this data is generally performed on a study-by-study case without any specific hypothesis of the location for SNPs that are predictive for the phenotype. We have designed a method whereby very large SNP lists (several gigabytes in size), combining several genotyping studies at once, can be sorted and traced back to their ultimate consequence in protein structure. Given a working hypothesis, researchers are able to easily search whole genome genotyping data for SNPs that link genetic locations …

Gambogic Acid Is A Tissue-Specific Proteasome Inhibitor In Vitro And In Vivo, Xiaofen Li, Shouting Liu, Hongbiao Huang, Ningning Liu, Chong Zhao, Siyan Liao, Changshan Yang, Yurong Liu, Canguo Zhao, Shujue Li, Xiaoyu Lu, Chunjiao Liu, Lixia Guan, Kai Zhao, Xiaoqing Shi, Wenbin Song, Ping Zhou, Xiaoxian Dong, Haiping Guo, Guanmei Wen, Change Zhang, Lili Jiang, Ningfang Ma, Bing Li, Shunqing Wang, Huo Tan, Xuejun Wang, Q. Ping Dou, Jinbao Lin

Oncology Faculty Publications

Gambogic acid (GA) is a natural compound derived from Chinese herbs that has been approved by the Chinese Food and Drug Administration for clinical trials in cancer patients; however, its molecular targets have not been thoroughly studied. Here, we report that GA inhibits tumor proteasome activity, with potency comparable to bortezomib but much less toxicity. First, GA acts as a prodrug and only gains proteasome-inhibitory function after being metabolized by intracellular CYP2E1. Second, GA-induced proteasome inhibition is a prerequisite for its cytotoxicity and anticancer effect without off-targets. Finally, because expression of the CYP2E1 gene is very high in tumor tissues …

Characterization Of High-Voltage-Activated Calcium Channels In Retinal Bipolar Cells, Qi Lu

Wayne State University Dissertations

Retinal bipolar cells, conveying visual information from photoreceptors to ganglion cells, segregate visual information into multiple parallel pathways through their diversified cell types and physiological properties. Voltage-gated Ca²⁺ channels could be particularly important underlying the diversified physiological properties of different BCs. In this dissertation, I investigated the high-voltage-activated (HVA) calcium current in retinal bipolar cells in mice. In the first part of my dissertation, I characterized multiple bipolar cell-expressing GFP and/or Cre transgenic mouse lines. In the second part of my dissertation, by performing whole-cell patch-clamp recordings, I examined the electrophysiological properties of HVA calcium currents among CBCs and …

Influence Of Mechanical Cues And The Extracellular Matrix On Cell Migration Patterns And The Proliferation Rates Of Cells, Tara Shane Alger

Wayne State University Theses

The mechanical environment of a cell and its tissue can impact multiple biological processes including development, wound healing, and metastasis. Specific cellular behaviors influenced by the mechanical microenvironment include differentiation, morphology, apoptosis, migration, and proliferation. In this thesis I have focused specifically on the effect of environmental stiffness and applied mechanical forces on cellular migration and proliferation, respectively. Using two different applications, both tailored to evaluate the mechanical forces alone on cellular behavior, I attempted to simulate the mechanical composition of the in vivo tissue microenvironments in vitro using polyacrylamide hydrogels. To test whether cells maintain a mechanical memory for …

Regulation Of Nuclear Localization Of The Sole Sumo-Conjugating Enzyme, Ubc9, Palak Sekhri

Wayne State University Theses

The covalent and reversible conjugation of small ubiquitin-like modifier (SUMO) proteins to hundreds of different cellular proteins is catalyzed by a cascade of enzymes including an E1-activating enzyme (SAE1/SAE2), an E2-conjugating enzyme (Ubc9) and multiple E3 ligases. As the only E2 enzyme for SUMO-conjugation, Ubc9 localizes mainly in the nucleus and plays an essential role in regulation of many cellular processes including cell cycle progression through mitosis, cell migration, genome stability, stress response, transcription, and nuclear transport in eukaryotic cells. It is hypothesized that the nuclear localization of Ubc9 is required for efficient sumoylation inside the nucleus because both the …

Optimization Of Labeling Techniques; Determination Of Best Parameter For Olfactory Mucosal Progenitor Cell Delivery And Study Of Effects Of Methylene Blue And Polyethylene Glycol In An Animal Model Of Spinal Cord Injury, Kiran Kumar Rokkappanavar

Wayne State University Theses

In the United States, the incidence of TSCI is about 40 per million persons per year, with approximately 250,000 living survivors of TSCI in the United States in July 2005. A number of human clinical trials (85) are ongoing using stem cells to evaluate the methods to reduce the injury following TBI. Among the stem cells, olfactory mucosal progenitor cells have several advantages with respect to ease of obtaining, fate of cells, etc. Our study was intended to optimize the labeling technique and to determine the best parameter to deliver the stem cells to the site of injury. We found …

The Xenopus Lefty (Xlefty) Prodomain Negatively Regulates Xlefty Activity And Is Necessary For Proper Xlefty Secretion, Adrian Amelio Vasquez

Wayne State University Theses

In vertebrates, the TGF #914; superfamily of secreted peptides are stringently regulated since they are responsible for multiple cell processes and behaviors that give rise to the proper patterning of embryonic tissues. Examples of members of the TGF #914; superfamily are Nodal and Lefty. Dysregulation of these proteins can lead to many diseases and developmental syndromes in humans.

Lefty functions by antagonizing Nodal an essential organizer signal that patterns dorsal mesoderm and the embryonic axes. Loss of Lefty expression results in excess Nodal signaling which has been shown to cause several perturbations including metastatic cancer. Although studies have clearly shown …

Defining The Mechanism Of Enhanced Cellular Invasion Induced By Mechanical Stimulation, Snehal Sunil Ozarkar

Wayne State University Theses

Metastasis is a multistep process driven by various biochemical and mechanical factors, which eventually leads to formation of secondary tumors. The tumor mass is surrounded by basement membrane (BM) and stroma made of various extracellular matrix (ECM) proteins. During metastasis tumor cells disseminate from the primary tumor, breach the BM, invade the stroma, travel through blood and lymph and colonize tissues distant from the primary tumor. Formation of secondary tumors by metastasis is a leading cause of death in cancer patients. Even though plenty of research has been focused on biochemical factors affecting metastasis, information on role of mechanical factors …

Studies Of Regulated Exocytosis From Neuroendocrine Cells, Madhurima Das

Wayne State University Theses

In this thesis we study cargo release and fusion pore dilation during calcium triggered exocytosis and the co-localization of calcium sensing proteins essential for exocytosis, in neuroendocrine cells.

Pancreatic beta cells secrete several hormones, the most studied one being insulin. C-peptide is a protein which is co-stored with and secreted from the same vesicles as insulin. It is found in the soluble phase unlike insulin, which is found in the dense core. The pancreatic beta cells also secrete the Chromogranin B (CgB) which is mostly found in the dense cores of secretory vesicles. In chapter 1, we found that CgB, …

Desensitization Of Basal Cell Carcinoma To The Anti-Tumoral Effect Of Vitamin D, Rawia Khasawneh

Wayne State University Dissertations

The relationship between Vitamin D and NMSC is not very clear. Most of human studies in literature in regards to vitamin D and NMSC are epidemiological which are inconsistent and inconclusive. Mechanistic studies about BCC are few, and mostly are animal models or cell cultures. The main goal for this study is to interpret the impact of vitamin D status on the onset and progression and possibly treatment of basal cell carcinoma. Three tissue samples were collected from BCC patients (Cancer, Proximal, and Distal), and from cancer-free individuals from southeast Michigan. This study design helps identifying alterations in the genetic …

The Transcriptional Regulation Of Flagellin-Induced Innate Protection Of The Cornea: Role Of Irf1 And Atf3, Gi Sang Yoon

Wayne State University Dissertations

Pre-exposure of the cornea to TLR5 ligand flagellin induces profound mucosal innate protection against infections by reprogramming gene expression. This study explored the flagellin-induced modifications of transcription factor expression and function, specifically of IRF1 and ATF3 in corneal epithelial cells to elucidate the transcriptional mechanisms underlying the protective function of flagellin on the cornea.

Initially we used Superarray to screen for transcription factors and identified Interferon Regulatory Factor (IRF) 1 and Activating Transcription Factor (ATF) 3 as the most drastically affected genes by flagellin pretreatment in P. aeruginosa challenged human corneal epithelial cells (CEC). However, flagellin pretreatment had opposite effects …

Acidic Pericellular Ph: Effects On Proteolysis And Gene Expression As Determined In 3d Models Of Breast Carcinoma, Jennifer M. Rothberg

Wayne State University Dissertations

Among the non-cellular microenvironmental factors that contribute to malignancy of solid tumors is an acidic peritumoral pH. The first objective was to determine if an acidic extracellular pH observed in vivo (i.e., pHe 6.8) affects the activity of proteases, such as cathepsin B, that contribute to degradation of collagen IV by tumor cells when grown in biologically relevant three-dimensional cultures. At pHe 6.8 there were increases in pericellular active cysteine cathepsins and in degradation of DQ-collagen IV, which was partially blocked by a cathepsin B inhibitor. Imaging probes for active cysteine cathepsins localized to tumors in vivo. The amount of …

Activation Of Amp-Activated Protein Kinase By 3,39-Diindolylmethane (Dim) Is Associated With Human Prostate Cancer Cell Death In Vitro And In Vivo, Di Chen, Sanjeev Banerjee, Qiuzhi C. Cui, Dejuan Kong, Fazlul H. Sarkar, Q. Ping Dou

Oncology Faculty Publications

There is a large body of scientific evidence suggesting that 3,39-Diindolylmethane (DIM), a compound derived from the digestion of indole-3-carbinol, which is abundant in cruciferous vegetables, harbors anti-tumor activity in vitro and in vivo. Accumulating evidence suggests that AMP-activated protein kinase (AMPK) plays an essential role in cellular energy homeostasis and tumor development and that targeting AMPK may be a promising therapeutic option for cancer treatment in the clinic. We previously reported that a formulated DIM (BR-DIM; hereafter referred as B-DIM) with higher bioavailability was able to induce apoptosis and inhibit cell growth, angiogenesis, and invasion of prostate cancer cells. …