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STUDIES OF NORSPERMIDINE UPTAKE IN DROSOPHILA SUGGEST THE EXISTENCE OF MULTIPLE POLYAMINE TRANSPORT PATHWAYS
Title
STUDIES OF NORSPERMIDINE UPTAKE IN DROSOPHILA SUGGEST THE EXISTENCE OF MULTIPLE POLYAMINE TRANSPORT PATHWAYS.
Creator
Dieffenbach, Michael, Von Kalm, Laurence, Teter, Kenneth, University of Central Florida
Abstract / Description
Polyamines are a class of essential nutrients involved in many basic cellular processes such as gene expression, cell proliferation, and apoptosis. Without polyamines, cell growth is delayed or halted. Cancerous cells require an abundance of polyamines through a combination of synthesis and transport from the extracellular environment. An FDA-approved drug, D,L-?-difluoromethylornithine (DFMO), blocks polyamine synthesis but is ineffective at inhibiting cell growth due to polyamine transport....
Show morePolyamines are a class of essential nutrients involved in many basic cellular processes such as gene expression, cell proliferation, and apoptosis. Without polyamines, cell growth is delayed or halted. Cancerous cells require an abundance of polyamines through a combination of synthesis and transport from the extracellular environment. An FDA-approved drug, D,L-?-difluoromethylornithine (DFMO), blocks polyamine synthesis but is ineffective at inhibiting cell growth due to polyamine transport. Thus, there is a need to develop drugs that inhibit polyamine transport to use in combination with DFMO. Surprisingly, little is known about the polyamine transport system in humans and other eukaryotes. Understanding the transport system would allow us to identify compounds that inhibit polyamine transport, which could then be used in tandem with DFMO to treat cancer. Our laboratory has identified one gene in Drosophila, called CG32000, as a component of this transport system, and numerous other candidate genes remain to be tested. To better characterize this system, this project investigated the ability of the Drosophila transport system to take up a toxic polyamine analogue called norspermidine, with the initial goal of developing a new screening method to find polyamine transport genes. My experiments have demonstrated significant differences in norspermidine uptake and toxicity between C. elegans and Drosophila which may imply a secondary polyamine transport system in higher eukaryotes. In the long term, it is hoped that this thesis will facilitate the development of more effective cancer medications by providing new information about the polyamine transport system.
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Date Issued
2018
Identifier
CFH2000294, ucf:45869
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFH2000294
A chemical and genetic approach to study the polyamine transport system in Drosophila
Title
A chemical and genetic approach to study the polyamine transport system in Drosophila.
Creator
Wang, Minpei, Vonkalm, Laurence, Phanstiel, Otto, Teter, Kenneth, Ballantyne, John, University of Central Florida
Abstract / Description
Polyamines are small cationic molecules that play important roles in most vital cellular processes including cell growth and proliferation, regulation of chromatin structure, translation and programmed cell death. Cellular polyamine pools are maintained by a balance between biosynthesis and transport (export and import). Increased polyamine biosynthesis activity and an active transport system are characteristics of many cancer cell lines, and polyamine depletion has been shown to be a viable...
Show morePolyamines are small cationic molecules that play important roles in most vital cellular processes including cell growth and proliferation, regulation of chromatin structure, translation and programmed cell death. Cellular polyamine pools are maintained by a balance between biosynthesis and transport (export and import). Increased polyamine biosynthesis activity and an active transport system are characteristics of many cancer cell lines, and polyamine depletion has been shown to be a viable anticancer strategy. Polyamine levels can be depleted by ?-difluoromethylornithine (DFMO), an inhibitor of the key polyamine biosynthesis enzyme ornithine decarboxylase. However, malignant cells often circumvent DFMO therapy by up-regulating polyamine import; therefore, there is a need to develop compounds that inhibit polyamine transport. Collectively, DFMO and polyamine transport inhibitors provide the basis for a combination therapy leading to effective intracellular polyamine depletion. Using a Drosophila leg imaginal disc model for polyamine transport, I studied three candidate transport inhibitors (Ant444, Trimer44 and Triamide44) for their ability to inhibit transport in the Drosophila model. Ant444 and Trimer44 effectively inhibited the uptake of the toxic polyamine analog Ant44 that gains entry to cells via the polyamine transport system. Ant444 and Trimer44 were also able to inhibit the import of exogenous polyamines into DFMO-treated imaginal discs. Triamide44 was an ineffective inhibitor, however a structurally redesigned compound, Triamide444, showed a 50-fold increase in transport inhibition and was comparable to Ant444 and Trimer44. Ant444 and Trimer44 showed differences in their relative abilities to block import of specific polyamines, and I therefore asked if a cocktail of these inhibitors would be more effective than either alone. My data show that a cocktail of polyamine transport inhibitors is more effective than single inhibitors when used in combination with DFMO, and suggests the existence of multiple polyamine transport systems. To further the development of effective transport inhibitors it is important to identify components of the transport system. The mechanism of polyamine transport in multicellular organisms including mammals is still unknown. Our laboratory has developed a simple assay to detect components of the transport system using RNAi knockdown and over-expression of candidate genes. However, the assay requires that animals live until the pupal stage of development. Pleiotropic effects of individual gene products following over-expression or knockdown may result in early developmental lethality for reasons unrelated to polyamine transport. Our assay is based on the GAL4/UAS system and involves the use of enhancers driving GAL4 expression (GAL4 driver). GAL4 in turn determines the expression level of UAS-candidate gene constructs (UAS responder). I reasoned that in some cases it might be possible to bypass early lethality by judicious choice of drivers that reduce responder expression, thus permitting survival to the pupal phase. To this end, I used five imaginal disc drivers (30A, 71B, 32B, 69B, and T80) as well as a ubiquitously expressed control driver to over-express and knockdown EGFR and components of the Rho signaling pathway. The relative strength of each driver was ranked, and I was able to demonstrate in principle that animals could survive to later stages of development in a manner that correlated with the relative strength of the driver. The approach I developed is broadly applicable to other studies of Drosophila development.To identify new components of the polyamine transport system I studied the role of proteoglycans in this process. The proteoglycan glypican-1 has been previously implicated in mammalian polyamine transport. In particular, the heparin sulfate side chains of glypican-1 appear to play an important role. In order to extend our knowledge of the role of proteoglycans in polyamine transport, I examined the role of the core proteoglycans perlecan and syndecan as well as genes encoding enzymes in the heparin sulfate and chondroitin sulfate biosynthetic pathways. I was able to confirm a role for glypican-1 in polyamine transport in imaginal discs but not in whole animals. This may indicate that glypican-1 is not required for polyamine uptake through the gut. Studies of genes encoding perlecan, syndecan and enzymes in the heparin sulfate and chondroitin sulfate biosynthetic pathways did not reveal a role for these genes in polyamine transport. These studies were conducted in whole animals and my data may reflect tissue-specific differences between the imaginal disc and gut transport systems where transport in imaginal discs is proteoglycan dependent and transport in the gut is not.
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Date Issued
2017
Identifier
CFE0007297, ucf:52162
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0007297
Design, Synthesis, and Biological Evaluation of Novel Polyamine Transport System Probes and their Application to Human Cancers
Title
Design, Synthesis, and Biological Evaluation of Novel Polyamine Transport System Probes and their Application to Human Cancers.
Creator
Muth, Aaron, Phanstiel, Otto, Ye, Jingdong, Elsheimer, Seth, Miles, Delbert, Vonkalm, Laurence, University of Central Florida
Abstract / Description
The mammalian polyamine transport system (PTS) has been of interest due to its roles in cancer and maintaining cellular homeostasis. Polyamines are essential growth factors which are tightly controlled via a balance of biosynthesis, metabolism, import, and export. This work focused on the development and biological testing of polyamine transport probes to help understand the molecular requirements of the PTS. This was mediated through the use of a CHO (PTS active) and CHO-MG* (PTS deficient)...
Show moreThe mammalian polyamine transport system (PTS) has been of interest due to its roles in cancer and maintaining cellular homeostasis. Polyamines are essential growth factors which are tightly controlled via a balance of biosynthesis, metabolism, import, and export. This work focused on the development and biological testing of polyamine transport probes to help understand the molecular requirements of the PTS. This was mediated through the use of a CHO (PTS active) and CHO-MG* (PTS deficient) screen, where compounds demonstrating high toxicity in CHO and low toxicity in CHO-MG* were considered PTS selective. The first chapter focused on the development of polyamine-based drugs which are both metabolically stable to polyamine oxidase (PAO) activity and are hyperselective for targeting the PTS. This approach was optimized by combining a di-substituted aryl design with terminal N-methylation of the appended polyamine chains to generate a new class of superior PTS agonists. The metabolic stability of these compounds was demonstrated in CHO and CHO-MG* in the presence and absence of a known PAO inhibitor, aminoguanidine (AG). Highly PTS selective compounds were then tested in the NCI-60 cell line screen to demonstrate the effectiveness of polyamine-based drugs in cancer therapy. During this screen, the MALME-3M (human melanoma) cell line was identified as being very sensitive to these PTS targeting drugs. Further studies using MALME-3M and its normal counterpart, MALME-3, showed excellent targeting of the cancer line over MALME-3. For example, The MeN44Nap44NMe compound showed 59-fold higher toxicity in MALME-3M over MALME-3.The second chapter focused on the development of potential polyamine transport inhibitors (PTIs) for use in combination therapy with ?-difluoromethylornithine (DFMO). This therapy is predicated upon reducing sustained polyamine depletion within cells by inhibiting both polyamine biosynthesis with DFMO and polyamine transport with the PTI ligand. Potential PTIs were identified by blocking the uptake of spermidine in DFMO-treated CHO and L3.6pl cells. Previous work has identified a tri-substituted polyamine-based design as an effective PTI. Low toxicity and a low Ki value in a L1210 screen were good predictors for PTI efficacy. The structural requirements for a potent PTI were explored by modulating the toxicity through the introduction of amide bonds, and also by determining the number and orientation of the polyamine messages (appended to an aryl core) required for efficient inhibition of polyamine uptake. These experiments showed that a tri-substituted design and a triamine message (homospermidine) appended was optimal for PTI potency. The final chapter focused on the development of Dihydromotuporamine C derivatives as non-toxic anti-metastatic agents. Dihydromotuporamine C demonstrated good anti-invasive properties with tumor cells. Derivatives were made in an effort to reduce the cytotoxicity of the parent and improve the anti-migration potency. The motuporamine derivatives all have a polyamine message (norspermidine or homospermidine) appended to make a macrocycle core, making them prime targets to evaluate as potential PTS ligands in the CHO and CHO-MG* screen. Each compound was also tested in the highly metastatic pancreatic cancer cell line L3.6pl to determine both its IC50 value and maximum tolerated dose (MTD). The anti-migration assay was performed at the lowest MTD obtained (0.6 (&)#181;M) in order to compare the series at the same non-toxic dose. The results suggested that as the N1-amine center was moved further from the macrocyclic ring, an increased ability to inhibit cell migration and reduced toxicity was observed. These collective findings provide new tools for cell biologists to modulate and target polyamine transport in mammalian cells. Future applications of these technologies include new cancer therapies which are cell-selective and inhibit the spread of tumors.
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Date Issued
2012
Identifier
CFE0004636, ucf:49895
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0004636
AN RNAI SCREEN TO IDENTIFY COMPONENTS OF A POLYAMINE TRANSPORT SYSTEM
Title
AN RNAI SCREEN TO IDENTIFY COMPONENTS OF A POLYAMINE TRANSPORT SYSTEM.
Creator
Foley, Adam J, Von Kalm, Laurence, University of Central Florida
Abstract / Description
Polyamines, specifically putrescine, spermidine, and spermine, are small cationic molecules found in all organisms. Cells can biosynthetically make these molecules, or alternatively, they can be transported from the extracellular environment. Malignant cells have been shown to require relatively high amounts of polyamines. There is a chemotherapeutic agent, DFMO, used to block the biosynthesis of polyamines. Many malignant cells can circumvent DFMO therapy by activating their transport system...
Show morePolyamines, specifically putrescine, spermidine, and spermine, are small cationic molecules found in all organisms. Cells can biosynthetically make these molecules, or alternatively, they can be transported from the extracellular environment. Malignant cells have been shown to require relatively high amounts of polyamines. There is a chemotherapeutic agent, DFMO, used to block the biosynthesis of polyamines. Many malignant cells can circumvent DFMO therapy by activating their transport system. A potential solution is to simultaneously block biosynthesis and transport of polyamines. However, little is known about the polyamine transport system in higher eukaryotes. This thesis aims to add to the basic biological understanding of the polyamine transport system, as well as contribute to our understanding of the way in which malignant cells are able to sustain rapid growth. This was done by screening six candidate genes believed to be involved in the polyamine transport system. These six genes were identified using various bioinformatics databases. They were screened using RNAi to knock down each gene of interest and by using an assay developed in our lab. One of the genes, RabX6, may play a possible role in the transport of putrescine.
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Date Issued
2017
Identifier
CFH2000187, ucf:46043
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFH2000187
POLYAMINE TRANSPORT INHIBITOR EFFECTS ON PANCREATIC CANCER PROLIFERATION CELLS IN VIVO
Title
POLYAMINE TRANSPORT INHIBITOR EFFECTS ON PANCREATIC CANCER PROLIFERATION CELLS IN VIVO.
Creator
Hogan, Frederick, Altomare, Deborah, University of Central Florida
Abstract / Description
Pancreatic cancer is a serious disease, one in which the survival rate over five years is less than 6%. (1) Often, malignant tumors will exhibit uncontrolled proliferation and it is postulated that they have high metabolic needs. One of the areas of interest in cancer metabolism is the unique need for large amounts of polyamines in order to sustain this uncontrolled proliferation. Polyamines are organic compounds that all cells need for proliferation and differentiation. (2) Cells obtain...
Show morePancreatic cancer is a serious disease, one in which the survival rate over five years is less than 6%. (1) Often, malignant tumors will exhibit uncontrolled proliferation and it is postulated that they have high metabolic needs. One of the areas of interest in cancer metabolism is the unique need for large amounts of polyamines in order to sustain this uncontrolled proliferation. Polyamines are organic compounds that all cells need for proliferation and differentiation. (2) Cells obtain polyamines by manufacture of them directly or obtain them from the environment through their transport across the cell membrane. When cancer cells have limited access to polyamines they enter apoptosis, or controlled cell death. In animal models of cancer, cellular apoptosis can be tracked by measuring tumor weights as well as histological methods. It is known that difluromethylornithine (DFMO), a polyamine synthesis inhibitor, has shown some success in reducing tumor weights. (3) Cells deprived of the ability to manufacture polyamines will resort to transporting existing polyamines from the environment for their use. (3) It is believed that a polyamine transport inhibitor (PTI) can be designed and used in conjunction with DFMO to completely deprive cancer cells of polyamines and increase rates of apoptosis in tumor cells. Through this study the interaction of DFMO with newly identified PTI will be analyzed. A mouse model with tumor cells in the pancreas will give a picture of how cancer cells react to DFMO / PTI in vivo. The findings allow us to postulate how targeted compounds interact with protein signaling pathways that may be important for regulating response to inhibitors of polyamine synthesis and/or transport.
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Date Issued
2015
Identifier
CFH0004749, ucf:45395
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFH0004749
The development of motuporamine derivatives and an investigation into their biological properties
Title
The development of motuporamine derivatives and an investigation into their biological properties.
Creator
Skruber, Kristen, Phanstiel, Otto, Teter, Kenneth, Vonkalm, Laurence, University of Central Florida
Abstract / Description
This project investigates the synthesis of a class of compounds derived from a marine-based natural product and probes how iterative changes to its structure affect its derivatives' biological efficacy. The compound class of interest are the motuporamines which were isolated from the sea sponge Xestospongia exigua collected off the coast of Motupore island in Papua, New Guinea. The compounds for this project are predicated upon dihydromotuporamine C (Motu33), the compound that has been shown...
Show moreThis project investigates the synthesis of a class of compounds derived from a marine-based natural product and probes how iterative changes to its structure affect its derivatives' biological efficacy. The compound class of interest are the motuporamines which were isolated from the sea sponge Xestospongia exigua collected off the coast of Motupore island in Papua, New Guinea. The compounds for this project are predicated upon dihydromotuporamine C (Motu33), the compound that has been shown to be both cytotoxic to MDA-MB231 breast carcinoma cells and has antimetastatic efficacy. The motuporamine scaffold contains a large fifteen-membered saturated macrocycle and an appended polyamine component. A series of Motu33 derivatives were synthesized and evaluated for their ability to target the polyamine transport system as well as inhibit cell migration of human pancreatic cancer cells in vitro. By altering the polyamine component of the system we attempted to build smart antimetastatic compounds which target the upregulated polyamine transport system of human pancreatic cancers and block their migration.
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Date Issued
2016
Identifier
CFE0006505, ucf:51390
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0006505
Novel Cytokine Signaling and Molecular Therapeutic Strategy in Pancreatic Cancer
Title
Novel Cytokine Signaling and Molecular Therapeutic Strategy in Pancreatic Cancer.
Creator
Gitto, Sarah, Altomare, Deborah, Khaled, Annette, Zhao, Jihe, Copik, Alicja, Masternak, Michal, University of Central Florida
Abstract / Description
Pancreatic ductal adenocarcinoma (PDAC) is highly chemo-resistant and has a five year survival rate of (
Show morePancreatic ductal adenocarcinoma (PDAC) is highly chemo-resistant and has a five year survival rate of (<)8%. Risk factors of pancreatic cancer, such as chronic pancreatitis, help to elicit a pro-tumor immune response, and highly fibrotic environment that promotes tumorigenesis. To study how chronic pancreatitis promotes cancer initiation, traditional KRasG12D mice and double mutant Akt1Myr/KrasG12D mice were used to model microenvironment changes. Akt1Myr/KrasG12D mice were more susceptible to chronic tissue damage, accelerated tumor development and metastatic disease. These mice exhibited histological changes consistent with immune cell privilege, where M2 macrophages and non-cytotoxic eosinophils were co-localized with fibrotic regions. IL-5 expression was up regulated in pancreatic cells undergoing acinar to ductal metaplasia and then diminished in advanced lesions. Tumor cells treated with IL-5 exhibit increased migration and activation through STAT5 signaling. Collectively, the results suggest that eosinophils, which are responsive to IL-5, are key mediators in the pancreatic environment subjected to chronic inflammation and injury.Current therapeutics fall short in increasing patient survival. There remains an urgent need for innovative treatments and thus we tested difluoromethylornithine (DFMO) in combination with a novel polyamine transport inhibitor, Trimer44NMe, against Gemcitabine-resistant PDAC cells. Prior clinical failures when targeting polyamine biosynthesis with DFMO monotherapy may be due to tumor escape via an undefined polyamine transport system. In pancreatic tumor cells DFMO alone and with Trimer44NMe significantly reduced PDAC cell viability by inducing apoptosis or cell cycle arrest. In vivo orthotopic PDAC growth with DFMO treatment resulted in decreased c-Myc expression, a readout of polyamine pathway dysfunction. Moreover, dual inhibition significantly prolonged survival of tumor-bearing mice, and increased M1 macrophage infiltration and reduced FoxP3 expression. Collectively, these studies demonstrate that targeting polyamine pathways in PDAC is a promising immunomodulating therapy that increases survival.
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Date Issued
2017
Identifier
CFE0007283, ucf:52168
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0007283