Alumni Dissertations and Theses

 
 

Alumni Dissertations and Theses

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  • PROTEASOME IMPAIRMENT, MITOCHONDRIA DYSFUNCTION, AND TAU PATHOLOGY IN ALZHEIMER DISEASE

    Author:
    QIAN HUANG
    Year of Dissertation:
    2012
    Program:
    Biochemistry
    Advisor:
    MARIA FIGUEIREDO-PEREIRA
    Abstract:

    Impairment of the ubiquitin/proteasome pathway is implicated in the pathogenesis of many neurodegenerative disorders, such as Alzheimer disease (AD). This is supported by detection of ubiquitinated protein aggregates in neurofibrillary tangles (NFTs) (Ross and Poirier, 2004) and reduction of proteasome activity in autopsied brains from AD patients (Keller et al., 2000). However, the upstream events leading to impairment of the ubiquitin/proteasome pathway are not fully understood. The ubiquitin/proteasome pathway is a highly regulated and efficient pathway that is critical for degradation of most intracellular proteins (Ciechanover, 2005). Ubiquitination, proteasome assembly and proteasome activity are energy dependent processes that require ATP binding and ATP hydrolysis (Li and Demartino, 2009). In neurons, most ATP is generated by mitochondria. Emerging evidence implicates mitochondrial dysfunction and energy deficit in many neurodegenerative disorders such as AD (Lin and Beal, 2006). There are no current treatments that are neuroprotective in the sense that they slow or prevent further neurodegeneration in AD. To provide new insights for the development of novel and more effective therapeutic strategies that prevent/stop neurodegeneration in AD, the MAJOR AIMS of these studies were: (1) Investigate underlying mechanisms that link mitochondrial dysfunction to impairment of the ubiquitin/proteasome pathway. (2) Evaluate the protective effect of elevating cAMP against an endogenous product of inflammation, i.e. prostaglandin J2 that affects the ubiquitin/proteasome pathway. In our studies we used rat E18 cerebral cortical neuronal cultures to investigate the pathogenesis of AD. For the FIRST AIM, we mimicked mitochondrial dysfunction by treating neurons with the following mitochondrial toxins: (1) oligomycin which inhibits ATP synthase, (2) rotenone which inhibits mitochondrial complex I, and (3) antimycin which inhibits mitochondrial complex III. We demonstrate that mitochondrial impairment affects the ubiquitin/proteasome pathway in the neurons via: (1) Reducing polyubiquitinated protein levels by inhibiting the first step of the ubiquitination cascade that is ATP-dependent: ubiquitin adenylation by the enzyme E1, thus blocking E1-E2 transthiolation. (2) Downregulating 26S proteasomes by promoting their disassembly. Notably, we show for the first time to our knowledge, that calpain activation is one of the factors that contributes to 26S proteasome disassembly by selectively cleaving one of its subunits, Rpn10. Calpain activation is associated with necrosis, an energy-independent cell death pathway activated upon mitochondrial dysfunction. We also demonstrate that calpain cleaves pro-caspase 3 to an inactive fragment, most likely to prevent apoptosis that is an energy-dependent cell death pathway. Furthermore, calpain cleaved the microtubule associated protein TAU that is the major component of neurofibrillary tangles in AD and other tauopathies. (3) Promoting a rise in 20S proteasomes, which degrade most oxidized proteins as well as intrinsically disordered proteins in a ubiquitin- and energy-independent manner. (4) Finally, we show that down-regulation of ubiquitinated proteins and 26S proteasomes with a concomitant increase in 20S proteasomes, occur upon acute (up to 16h) and long-term (up to seven days) mitochondrial dysfunction in the neurons. Overall, these data support the notion that upon mitochondrial dysfunction, neurons initiate a series of mechanisms that converge to optimize unregulated and energy-independent turnover of randomly unfolded oxidized proteins by 20S proteasomes. This adaptive response to energy deficiency may be suitable for short-term periods. However, if maintained chronically it may lead to neurodegeneration, as regulated protein degradation by the ubiquitin/proteasome pathway is essential for neuronal survival. For the SECOND AIM, we treated rat E18 cerebral cortical neurons with prostaglandin J2 (PGJ2), an endogenous product of inflammation that affects mitochondrial function, and inhibits the 26S proteasome by causing its disassembly. We compared the effects of PGJ2 with those of the specific and irreversible proteasome inhibitor epoxomicin. We show that treatment with both drugs induces caspase-activation, TAU cleavage at Asp421 (deltaTAU), TAU and Ub-protein aggregation, and neuronal death. Truncation of TAU at Asp421 (deltaTAU) by caspases is an early event in AD tangle pathology. In addition, deltaTAU is detected in neurofibrillary tangles (NFTs) indicating that the apoptotic cascade is involved in NFT formation. To prevent these deleterious events associated with proteasome inhibition, we tested two drugs that elevate intracellular cAMP: dibutyryl-cAMP (db-cAMP) and the lipophilic peptide PACAP27 that increases intracellular cAMP levels by binding to its seven transmembrane G-coupled receptor PAC1R. Previous studies from our lab showed that a single dose of db-cAMP or PACAP27, blocked caspase-activation and deltaTAU upon short-term (up to 8h) treatment with PGJ2. One single dose of these cAMP-elevating drugs however, failed to protect against longer (24h) treatments with PGJ2. In our current studies, we decided to increase the number of drug treatments to improve protection. We established that three doses of either db-cAMP or PACAP27 successfully diminished caspase-activation, deltaTAU and loss of cell viability promoted by long-term (24h) incubations, as long as these changes were mild. Based on these studies, we propose that targeting cAMP/PKA to boost proteasome activity in a sustainable manner, could offer an effective approach to avoid early accumulation of ubiquitinated proteins and later caspase-activation and TAU cleavage, possibly preventing/ delaying neurodegeneration in AD. In summary, our data address two distinct death pathways in neurons that are associated with mitochondria and proteasome impairment: necrosis via calpain activation and apoptosis via caspase activation. We show that on one hand, mitochondria-dependent energy deficit affects ubiquitination and 26S proteasomes, and promotes cleavage of pro-caspase 3 and TAU via calpain activation. These events are associated with necrotic neuronal death. On the other hand, proteasome impairment induces the accumulation of ubiquitinated proteins and promotes TAU cleavage via caspase 3 activation. These events are associated with neuronal apoptotic death. The dual vulnerability of neurons to calpain- and caspase-mediated cell death needs to be taken into consideration when considering therapeutic approaches to prevent neuronal demise in AD and other chronic neurodegenerative diseases associated with proteasome and mitochondria dysfunction.

  • Role of Fibroblast Growth Factor Homologous Factors in Excitability of Hippocampal Neurons

    Author:
    Xiao Huang
    Year of Dissertation:
    2009
    Program:
    Biochemistry
    Advisor:
    Mitchell Goldfarb
    Abstract:

    Fibroblast growth factor homologous factors (FHFs) are a family of vertebrate neuronal proteins which function in manners distinct from fibroblast growth factors (FGFs). While bearing substantial sequence and structural homology to FGFs, FHFs reside intracellularly and bind targets unrelated to FGF receptors, which include voltage-gated sodium channels (Navs). FHFs have been shown to control excitability of cerebellar granule cells through modulation of channel inactivation. Since action potentials are initiated at axon initial segment (AIS), we suspected that at least some FHF isoforms would reside at AIS in association with Navs. As expected, a broad repertoire of FHF isoforms colocalizes with Navs at the AIS of cultured hippocampal neurons. Moreover, together with other studies in our laboratory, the present study shows that FHF "b" isoform associates with AIS to a far lesser extent than "a" isoform, demonstrating that there is isoform specificity in FHF targeting. AIS targeting of an FHF requires the protein's channel binding surface, as a mutant derivative of FHF2a deficient for channel binding is also deficient in AIS targeting. The association of FHFs with Navs at AIS suggests that FHFs may modulate channel physiology, thereby controlling the intrinsic excitability of hippocampal neurons in a manner similar to that which has been described for cerebellar granule neurons of Fhf1-/-Fhf4-/- mice. Moreover, real time PCR shows that FHF2 is the most abundant of the FHFs expressed in hippocampal neurons. The requirement of FHF2 for hippocampal neuron excitability was analyzed using RNAi in conjunction with electrophysiological recordings. These studies have demonstrated that knockdown of fhf2 in hippocampal neurons derived from Fhf1-/- mice impairs excitability, with less maximum spike frequency and elevated voltage thresholds for spike induction. Sodium channel inactivation parameters are altered in Fhf1-/-Fhf4-/- granule neurons. In accordance, the colocalization and physical interaction of FHFs with Navs in hippocampal neurons suggest that excitability deficits could reflect altered channel physiology in cells lacking FHF1 and FHF2 function. These are the first data to indicate a role for FHF2 in neuronal excitability. These data suggest a widespread role for FHFs in the control of excitability across the central nervous system (CNS).

  • Role of Fibroblast Growth Factor Homologous Factors in Excitability of Hippocampal Neurons

    Author:
    Xiao Huang
    Year of Dissertation:
    2009
    Program:
    Biochemistry
    Advisor:
    Mitchell Goldfarb
    Abstract:

    Fibroblast growth factor homologous factors (FHFs) are a family of vertebrate neuronal proteins which function in manners distinct from fibroblast growth factors (FGFs). While bearing substantial sequence and structural homology to FGFs, FHFs reside intracellularly and bind targets unrelated to FGF receptors, which include voltage-gated sodium channels (Navs). FHFs have been shown to control excitability of cerebellar granule cells through modulation of channel inactivation. Since action potentials are initiated at axon initial segment (AIS), we suspected that at least some FHF isoforms would reside at AIS in association with Navs. As expected, a broad repertoire of FHF isoforms colocalizes with Navs at the AIS of cultured hippocampal neurons. Moreover, together with other studies in our laboratory, the present study shows that FHF "b" isoform associates with AIS to a far lesser extent than "a" isoform, demonstrating that there is isoform specificity in FHF targeting. AIS targeting of an FHF requires the protein's channel binding surface, as a mutant derivative of FHF2a deficient for channel binding is also deficient in AIS targeting. The association of FHFs with Navs at AIS suggests that FHFs may modulate channel physiology, thereby controlling the intrinsic excitability of hippocampal neurons in a manner similar to that which has been described for cerebellar granule neurons of Fhf1-/-Fhf4-/- mice. Moreover, real time PCR shows that FHF2 is the most abundant of the FHFs expressed in hippocampal neurons. The requirement of FHF2 for hippocampal neuron excitability was analyzed using RNAi in conjunction with electrophysiological recordings. These studies have demonstrated that knockdown of fhf2 in hippocampal neurons derived from Fhf1-/- mice impairs excitability, with less maximum spike frequency and elevated voltage thresholds for spike induction. Sodium channel inactivation parameters are altered in Fhf1-/-Fhf4-/- granule neurons. In accordance, the colocalization and physical interaction of FHFs with Navs in hippocampal neurons suggest that excitability deficits could reflect altered channel physiology in cells lacking FHF1 and FHF2 function. These are the first data to indicate a role for FHF2 in neuronal excitability. These data suggest a widespread role for FHFs in the control of excitability across the central nervous system (CNS).

  • Uncovering hidden potential of natural products

    Author:
    Maria Iacovidou
    Year of Dissertation:
    2010
    Program:
    Biochemistry
    Advisor:
    Akira Kawamura
    Abstract:

    Herbs and soil bacteria have been arguably the most important sources of therapeutic agents throughout human history. Numerous bioactive compounds have been isolated and characterized from these classical sources of natural products. Because of the long history of research on herbs and soil bacteria, they are sometimes perceived as “exhausted” sources of secondary metabolites. These classical sources of natural products, however, can still include compounds with previously overlooked chemical and biological properties. The unifying theme of this thesis study is to uncover such hidden potential of natural products. To this end, these classical sources of secondary metabolites have been examined from new angles. The first two chapters of this thesis describe the examination of oriental herbal formulations with biomarker–guided screening, which has been established recently in our group. In this method, natural products are screened based on their ability to modulate the expression of mRNA biomarkers, which are the genes associated with therapeutic effects of herbal medicine. Importantly, herbal formulations have never been screened in this manner. Thus, biomarker–guided screening can serve as a powerful approach to uncover hidden therapeutic potential of natural products. Chapter 1 describes the biomarker–guided screening of a blood cleansing herbal formulation called Toki–shakuyaku–san (TSS). This formulation has long been used to alleviate various disorders associated with poor blood circulation. Our screening revealed that a simple polyacetylene compound, (6E,12E)–tetradecadiene–8,10–diyne–1,3–diol diacetate (TDEYA), which has been considered as an “inactive” precursor of other metabolites, regulates the expression of genes associated with blood coagulation, such as COX–2, SerpinB2, and perlecan. Identification of this polyacetylene compound is important because it may open a new paradigm of treating circulation disorders through manipulation of gene expression by small molecules. Chapter 2 describes the biomarker-guided screening of another herbal formulation, Juzen–taiho–to (JTT), which is widely known as “immune booster” formulation. In fact, JTT is clinically used in Japan to reinvigorate the immune system of cancer patients undergoing chemotherapy and radiation therapy. It is believed that many beneficial effects of JTT are mediated by monocytes and macrophages. Key monocyte–stimulants in JTT have been difficult to identify despite many years of intensive studies by us and others. However, JTT biomarker–guided screening identified β–glucosylceramides (cerebrosides) as the first group of monocyte–stimulants in JTT. This discovery is an important first step to gain mechanistic insights into the clinically tested immunostimulatory effects of JTT. Finally, chapter 3 describes the screening of secondary metabolites from culturable bacteria from soil samples collected throughout the State of New York. While traditional screenings of secondary metabolites in soil bacteria focus on the search for new antibiotics, our screening looked for a compound with structural complexity which provides us with an access to the pharmacological spaces that are difficult to reach through organic synthesis. This is an important new goal of the screening of bacterial metabolites, because complex molecules of bacterial origin can now be produced from simple carbon sources through metabolic engineering. This study isolated and structurally characterized cyclooctatin B (COB), a new diterpene with a fused 5–8–5 ring system and unique U–shaped conformation. Our preliminary analyses suggest that COB can undergo remarkably elegant cascade reactions when its environment turns mildly acidic. Thus, COB may constitute a new class of pH–sensing bacterial metabolites with a variety of chemical, pharmaceutical, and engineering applications.

  • Uncovering hidden potential of natural products

    Author:
    Maria Iacovidou
    Year of Dissertation:
    2010
    Program:
    Biochemistry
    Advisor:
    Akira Kawamura
    Abstract:

    Herbs and soil bacteria have been arguably the most important sources of therapeutic agents throughout human history. Numerous bioactive compounds have been isolated and characterized from these classical sources of natural products. Because of the long history of research on herbs and soil bacteria, they are sometimes perceived as “exhausted” sources of secondary metabolites. These classical sources of natural products, however, can still include compounds with previously overlooked chemical and biological properties. The unifying theme of this thesis study is to uncover such hidden potential of natural products. To this end, these classical sources of secondary metabolites have been examined from new angles. The first two chapters of this thesis describe the examination of oriental herbal formulations with biomarker–guided screening, which has been established recently in our group. In this method, natural products are screened based on their ability to modulate the expression of mRNA biomarkers, which are the genes associated with therapeutic effects of herbal medicine. Importantly, herbal formulations have never been screened in this manner. Thus, biomarker–guided screening can serve as a powerful approach to uncover hidden therapeutic potential of natural products. Chapter 1 describes the biomarker–guided screening of a blood cleansing herbal formulation called Toki–shakuyaku–san (TSS). This formulation has long been used to alleviate various disorders associated with poor blood circulation. Our screening revealed that a simple polyacetylene compound, (6E,12E)–tetradecadiene–8,10–diyne–1,3–diol diacetate (TDEYA), which has been considered as an “inactive” precursor of other metabolites, regulates the expression of genes associated with blood coagulation, such as COX–2, SerpinB2, and perlecan. Identification of this polyacetylene compound is important because it may open a new paradigm of treating circulation disorders through manipulation of gene expression by small molecules. Chapter 2 describes the biomarker-guided screening of another herbal formulation, Juzen–taiho–to (JTT), which is widely known as “immune booster” formulation. In fact, JTT is clinically used in Japan to reinvigorate the immune system of cancer patients undergoing chemotherapy and radiation therapy. It is believed that many beneficial effects of JTT are mediated by monocytes and macrophages. Key monocyte–stimulants in JTT have been difficult to identify despite many years of intensive studies by us and others. However, JTT biomarker–guided screening identified β–glucosylceramides (cerebrosides) as the first group of monocyte–stimulants in JTT. This discovery is an important first step to gain mechanistic insights into the clinically tested immunostimulatory effects of JTT. Finally, chapter 3 describes the screening of secondary metabolites from culturable bacteria from soil samples collected throughout the State of New York. While traditional screenings of secondary metabolites in soil bacteria focus on the search for new antibiotics, our screening looked for a compound with structural complexity which provides us with an access to the pharmacological spaces that are difficult to reach through organic synthesis. This is an important new goal of the screening of bacterial metabolites, because complex molecules of bacterial origin can now be produced from simple carbon sources through metabolic engineering. This study isolated and structurally characterized cyclooctatin B (COB), a new diterpene with a fused 5–8–5 ring system and unique U–shaped conformation. Our preliminary analyses suggest that COB can undergo remarkably elegant cascade reactions when its environment turns mildly acidic. Thus, COB may constitute a new class of pH–sensing bacterial metabolites with a variety of chemical, pharmaceutical, and engineering applications.

  • Peptide Directed 3D Assembly of Nanoparticles through Biomolecular Interaction

    Author:
    Prerna Kaur
    Year of Dissertation:
    2012
    Program:
    Biochemistry
    Advisor:
    Hiroshi Matsui
    Abstract:

    The current challenge of the `bottom up' process is the programmed self-assembly of nanoscale building blocks into complex and larger-scale superstructures with unique properties that can be integrated as components in solar cells, microelectronics, meta materials, catalysis, and sensors. Recent trends in the complexity of device design demand the fabrication of three-dimensional (3D) superstructures from multi-nanomaterial components in precise configurations. Bio mimetic assembly is an emerging technique for building hybrid materials because living organisms are efficient, inexpensive, and environmentally benign material generators, allowing low temperature fabrication. Using this approach, a novel peptide-directed nanomaterial assembly technology based on bio molecular interaction of streptavidin and biotin is presented for assembling nanomaterials with peptides for the construction of 3D peptide-inorganic superlattices with defined 3D shape. We took advantage of robust natural collagen triple-helix peptides and used them as nanowire building blocks for 3D peptide- gold nanoparticles superlattice generation. The type of 3D peptide superlattice assembly with hybrid NP building blocks described herein shows potential for the fabrication of complex functional device which demands precise long-range arrangement and periodicity of NPs.

  • An Investigation on the Application of Mass Spectrometry Protein Footprinting Technique to Study Interactions of Collagen and Collagen Receptors

    Author:
    Michele Kirchner
    Year of Dissertation:
    2013
    Program:
    Biochemistry
    Advisor:
    Yujia Xu
    Abstract:

    Collagen is one of the most abundant proteins in the human body. There are many different types of collagen; they interact with an assortment of other proteins in the extracellular matrix, and with collagen receptors on the surfaces of cells. In this study, oxidative protein footprinting using Fenton chemistry followed by mass spectrometry was explored as a method to investigate collagen interactions. A known collagen binding protein, the A3 domain of the von Willebrand Factor protein (vWF A3), was chosen as a model. This protein association is the first critical step in blood clotting, and has been the target for the development of new drugs to treat von Willebrand disease, a bleeding disorder. Mass spectrometry protein footprinting could potentially aid in these endeavors. In order to accomplish this objective, original studies using Fenton chemistry labeling of triple helical peptides, and mass spectrometry collagen sequencing studies were carried out. The von Willebrand Factor A3 protein was produced from an Escherichia coli expression system. This work discovered many challenges that have to be overcome for the successful approach of mass spectrometry oxidative protein footprinting by Fenton chemistry to investigate full chain collagen interactions. Additionally, the mix of variable post translational modifications of collagen makes the identification of binding sites by footprinting unreliable. Even sequencing collagen proves problematic because of the many post translational modifications. Despite these setbacks, we did; however, find that the von Willebrand Factor A3 protein binds to a triple helix conformation lacking any of the presumed necessary hydroxyproline sites. We were also able to identify regions in full chain collagen where the oxidation content varied from what was expected, assuming all the prolines in the Y position were hydroxylated.

  • Characterization of Mitochondrial DNA Heteroplasmy at Five Hotspots within the HVI Region of Post-Mortem, Formalin Fixed Paraffinized Human Liver Cells

    Author:
    Jason Kolowski
    Year of Dissertation:
    2012
    Program:
    Biochemistry
    Advisor:
    Margaret Wallace
    Abstract:

    Within the field of mitochondrial DNA (mtDNA) analysis, heteroplasmy is a widely recognized and yet poorly-understood event. Heteroplasmy is defined as the presence of more than one mitochondrial genome within a tissue sample from a single individual, such that the mtDNA sequence shows the presence of a mixed base or regions of homologous bases that vary in length. Due to the highly conserved nature of the mitochondrial genome, these heteroplasmic events occur at a variety of well-documented hotspots, a majority of which occur within the hypervariable control region that flanks the origin of replication. This control region is the same area that is tested in forensic mtDNA analysis, and is the most useful for establishing the link between evidentiary samples and maternally-related individuals due to the polymorphisms that accumulate in this region. However, when heteroplasmic events are uncovered in forensic mtDNA analysis, issues arise due to the lack of clear understanding of the origin of heteroplasmy. The occurrence of mtDNA heteroplasmy between different tissues within a single individual has been well-established, and heteroplasmic events have been shown to increase with age. However, what is presently unclear is whether or not mtDNA heteroplasmy exists within a single cell when heteroplasmy is present within a tissue. In this regard, three possibilities exist; 1) a single cell contains a solitary pool of mtDNA genomes (defined as homoplastic), and heteroplasmy exists as a mixture of different homoplastic cells within tissue, 2) a single cell contains a mixture of multiple mtDNA genomes, and heteroplasmy is present within a single cell, or 3) heteroplasmic tissue contains a mixture of homoplastic and heteroplasmic cells due to random cellular distribution throughout the tissue. To investigate this question, laser dissection microscopy will be used to isolate individual cells from a tissue sample with known mtDNA heteroplasmy. Typing of single nucleotide polymorphisms at specific hotspots within the HVI region will then be done to detect possible mtDNA heteroplasmy within a single cell.

  • Strategic targeting of curcumin to eliminate brain tumors

    Author:
    Phyllis Langone
    Year of Dissertation:
    2011
    Program:
    Biochemistry
    Advisor:
    Probal Banerjee
    Abstract:

    Glioblastoma, the most common form of primary brain cancer, is highly aggressive and associated with very poor prognosis. Curcumin (or diferuloylmethane), a natural molecule, which is not toxic to normal tissue, has been shown to inhibit proliferation, induce apoptosis and inhibit angiogenesis and metastasis in a wide range of cancer cells. However, the effective delivery of curcumin to cancers presents a problem because curcumin is poorly soluble in water and metabolizes quickly. Our preliminary work has established that solubilized curcumin can cross the blood-brain barrier and is harmless to normal brain cells, that solubilized curcumin blocks brain tumor formation when introduced by injection into the blood or directly into the brain, and that it markedly decreases cell viability in several cell lines, including murine melanoma B16F10 and murine glioblastoma GL261. Targeted drug delivery is frequently used to deliver drugs selectively and at high concentrations to cancer tissue. Antibody-mediated targeting, in addition to delivering drugs selectively, serves to increase the water solubility of attached drugs. We postulated that antibody-mediated targeting would be an effective means of eliminating brain tumors. Nonetheless, a number of structural features had to be carefully considered. Curcumin has several functional groups, which potentially can be used to target the molecule to cancer cells; however, curcumin's functional groups have been shown to be critical for its anticancer activity. With this in mind, after weighing different options, we synthetically modified curcumin at its phenolic hydroxyl position to enable the formation of a cleavable antibody attachment. Intracellular hydrolysis of an ester bond returns curcumin to its original state after its delivery into target cells. Murine infiltrating melanoma (B16F10) and primary glioblastoma (GL261) brain tumor models were utilized. We created two adducts, curcumin-MUC18 for targeting to B16F10 cells and curcumin-CD68 for targeting to GL261 cells. Our studies show that both adducts are highly effective at eliminating B16F10 and GL261 cancer cells in vitro, and that these adducts destroy cancer cells at far lower concentrations than does free curcumin. Our molecular analyses show that, in GL261 cells, curcumin causes a dramatic increase in caspase 3/7 activity and suppression of tumor-promoting proteins NF-κB, Akt1, VEGF, cyclin D1, and BclXL. We show in GL261 cells that overexpressed NF-κB is protective against curcumin treatment. Lastly, we show that animals implanted with B16F10 or GL261 cells receiving targeted curcumin treatment live longer and have significantly reduced tumor size.

  • Strategic targeting of curcumin to eliminate brain tumors

    Author:
    Phyllis Langone
    Year of Dissertation:
    2011
    Program:
    Biochemistry
    Advisor:
    Probal Banerjee
    Abstract:

    Glioblastoma, the most common form of primary brain cancer, is highly aggressive and associated with very poor prognosis. Curcumin (or diferuloylmethane), a natural molecule, which is not toxic to normal tissue, has been shown to inhibit proliferation, induce apoptosis and inhibit angiogenesis and metastasis in a wide range of cancer cells. However, the effective delivery of curcumin to cancers presents a problem because curcumin is poorly soluble in water and metabolizes quickly. Our preliminary work has established that solubilized curcumin can cross the blood-brain barrier and is harmless to normal brain cells, that solubilized curcumin blocks brain tumor formation when introduced by injection into the blood or directly into the brain, and that it markedly decreases cell viability in several cell lines, including murine melanoma B16F10 and murine glioblastoma GL261. Targeted drug delivery is frequently used to deliver drugs selectively and at high concentrations to cancer tissue. Antibody-mediated targeting, in addition to delivering drugs selectively, serves to increase the water solubility of attached drugs. We postulated that antibody-mediated targeting would be an effective means of eliminating brain tumors. Nonetheless, a number of structural features had to be carefully considered. Curcumin has several functional groups, which potentially can be used to target the molecule to cancer cells; however, curcumin's functional groups have been shown to be critical for its anticancer activity. With this in mind, after weighing different options, we synthetically modified curcumin at its phenolic hydroxyl position to enable the formation of a cleavable antibody attachment. Intracellular hydrolysis of an ester bond returns curcumin to its original state after its delivery into target cells. Murine infiltrating melanoma (B16F10) and primary glioblastoma (GL261) brain tumor models were utilized. We created two adducts, curcumin-MUC18 for targeting to B16F10 cells and curcumin-CD68 for targeting to GL261 cells. Our studies show that both adducts are highly effective at eliminating B16F10 and GL261 cancer cells in vitro, and that these adducts destroy cancer cells at far lower concentrations than does free curcumin. Our molecular analyses show that, in GL261 cells, curcumin causes a dramatic increase in caspase 3/7 activity and suppression of tumor-promoting proteins NF-κB, Akt1, VEGF, cyclin D1, and BclXL. We show in GL261 cells that overexpressed NF-κB is protective against curcumin treatment. Lastly, we show that animals implanted with B16F10 or GL261 cells receiving targeted curcumin treatment live longer and have significantly reduced tumor size.