Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 156-169) Also available in print.

Thesis (Ph.D. in Cell and Developmental Biology) -- University of Colorado, 2005. / Typescript. Includes bibliographical references (leaves 155-180). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (M.Sc. (Biochemistry)) -- University of Limpopo, 2019 / Survivin is the smallest and a well-studied member of the inhibitors of apoptosis proteins (IAPs) family, which is involved in the regulation of cell division, inhibition of both caspasedependent and -independent apoptosis in cancer cells and promotion of angiogenesis. Survivin is detectable during embryonic and foetal development but is undetectable in normal adult tissues. It is, however, expressed in transformed cell lines as well as in most common types of human cancers. Regulation of survivin remains poorly understood, and the discovery of the regulatory biomolecules, microRNAs (MiRs) present an interesting opportunity to investigate the regulation of this protein and its variants in cancers, especially breast cancer. Additionally, the expression of the survivin splice variants during cell cycle progression and apoptosis is not fully understood. The aims of this study were to investigate the role of arsenic trioxide on the expression of survivin splice variants and their specific microRNAs during cell cycle progression and apoptosis in human breast cancer MCF-7 cells. The study also aimed at ascertaining the toxicity and efficacy of using coal fly ash-derived β-cyclodextrin carbon nanospheres to deliver arsenic trioxide into the MCF-7 cells. Carbon nanospheres (CNSs) were synthesised using a chemical vapour deposition method while arsenic trioxide was deposited using wet impregnation method to form the arsenic trioxide-β-cyclodextrin carbon nanospheres (ATO-β-cyclodextrin-CNSs). The formation of the CNSs and the loading of arsenic trioxide to CNSs were confirmed using scanning electron microscopy/energy dispersive X-ray detection (SEM-EDX). The in vitro cytotoxicity effect of the β-cyclodextrin carbon nanospheres (CNSs), arsenic trioxide and arsenic trioxide-β-cyclodextrin CNSs against KMST-6 and MCF-7 cells was analysed using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide (MTT) Assay, Muse® Count and Viability Assay and light/fluorescence microscopy. Cellular apoptosis, cell cycle analysis, Multi-Caspase activation, mitochondrial membrane potential, MAPK activation and PI3K activation were analysed using the Muse® Cell Analyser. Polymerase Chain Reaction (PCR) and Immunohistochemistry were used to analyse survivin mRNA variants and protein expression, respectively. The survivin specific MiRs were predicted using both bioinformatics platforms and literature surveys. In order to understand the applicability of delivering arsenic trioxide for the treatment of breast cancer, skin fibroblast (KMST-6) and MCF-7 cells were exposed to β-cyclodextrin CNSs. The novel β-cyclodextrin CNSs did not show any cytotoxic effect on the KMST-6 cells but demonstrated such activity against the MCF-7 cells. More so, arsenic trioxide-βcyclodextrin CNSs were found to significantly reduce the viability of the MCF-7 cells and were shown to inhibit their cell growth through the induction of apoptosis. The MTT Assay results revealed arsenic trioxide inhibited the growth of the MCF-7 cells in a concentration-dependent manner. The Muse® Cell Analyser showed that arsenic trioxide induced G2/M cell cycle arrest and promoted cellular apoptosis without any damage to the mitochondrial membrane of MCF-7 cells. Furthermore, arsenic trioxide also deactivated two survival pathways, Mitogen-Activated Protein Kinase (MAPK) and Phosphoinositide 3-Kinase (PI3K) signalling pathways in MCF-7 cells. The deactivation of the two pathways was shown to be accompanied by the upregulation of survivin 3α during arsenic trioxide-induced G2/M cell cycle arrest and apoptosis. Survivin 2B was found to be upregulated only during arsenic trioxide-induced G2/M cell cycle arrest, but downregulated during arsenic trioxide-induced apoptosis. However, wild-type survivin was highly expressed in untreated MCF-7 cells, but the expression was upregulated during arsenic trioxide-induced G2/M cell cycle arrest and was downregulated during arsenic trioxide-induced apoptosis. Survivin variant ΔEx3 was undetected in both untreated and treated MCF-7 cells. Survivin 2α was upregulated during arsenic trioxideinduced apoptosis whereas, survivin 3B was only detected in the untreated MCF-7 cells. Additionally, survivin proteins were localised in both the nuclei and cytoplasm in MCF-7 cells and highly upregulated during arsenic trioxide-induced G2/M cell cycle arrest, which can be attributed to the upregulation of survivin-2B. Using TargetScan, MIRD and mirTarbase, a few MiRs were identified and confirmed to target wild-type survivin, survivin 2B and survivin ΔEx3. These include the MiR-542-3p and MiR-335-5p, which are both upregulated during apoptosis and MiR-218-5p, which is upregulated during cell arrest. MiR-218-5p targets survivin 2B, which was upregulated during G2M cell cycle arrest. The fly ash-derived CNSs can be used to deliver arsenic trioxide for therapeutic purposes, especially against breast cancer. Most importantly, these nanoparticles induced typical apoptotic characteristics in breast cancer MCF-7 cells. Arsenic trioxide can be used as therapeutic target for breast cancer treatment and nanotechnology can be used for its delivery. This study provided the first evidence that novel survivin 2B splice variant may be involved in the regulation of arsenic trioxide-induced G2/M cell cycle arrest only. This splice variant can therefore, be targeted for therapeutic purposes against Luminal A breast cancer cells

Programmed cell death (apoptosis) is the most common mechanism of cell death in eukaryotes. The ability of cancer cells to evade and inhibit apoptosis has become a hallmark feature of cancer. This is accomplished through a family of proteins known as the inhibitor of apoptosis proteins (IAPs). X-Linked inhibitor of apoptosis protein (XIAP) is one of the best characterized IAPs. XIAP suppresses apoptosis by forming complexes with cysteine-aspartic proteases (caspase), through one of its baculovirus IAP repeat (BIR) domains. Its activity is endogenously antagonized by a second mitochondria derived activator of caspase (Smac). The anti-apoptotic behaviour of XIAP and the critical role it plays in the apoptotic program makes the Smac-XIAP interaction an important drug target. To this end, our laboratory is interested in synthesizing biologically related Smac mimetics which can induce apoptosis in a MDA-MB-231 cell line. Efforts have focused on (1) understanding BIR domain binding sites which allow for this interaction, and (2) the design and synthesis of molecules which are much more effective at inducing apoptosis compared to other well known analogues. Through the synthesis and evaluation of various divalent Smac mimetics we have been able to support the hypothesis that the likely binding site on XIAP is the BIR3 domain. As well, through the synthesis of a library of novel compounds, as described in the thesis, we have been able to assess the nature of the linker which joins the two tetrapeptide units. In our effort to understand which domains Smac binds with, various divalent analogues were synthesized containing MeAVPI-linker-IPVMeA (forward-reverse) and MeAVPI-linker-MeAVPI (forward-forward) sequence, which incorporated linkers with varying degrees of flexibility. We hypothesized that the forward-forward divalent mimetics would have decreased activity compared to the peptides synthesized in a forward-reverse fashion. Lastly, information gathered from structure activity relationship (SAR) studies have shown that substituting the lysine (P2) and isoleucine residues (P4) in the AVPI protein can create more potent inducers of apoptosis than its native AVPI sequence. As one of the most potent Smac mimetic that has been previously made known contains an alkyne bridge at P2 and a large hydrophobic moiety at P4, we hypothesized that similar Smac mimetics containing a propargyl glycine residue at P2 and a bulky hydrophobic moiety at P4 will be much more potent in inducing apoptosis.

Programmed cell death (apoptosis) is the most common mechanism of cell death in eukaryotes. The ability of cancer cells to evade and inhibit apoptosis has become a hallmark feature of cancer. This is accomplished through a family of proteins known as the inhibitor of apoptosis proteins (IAPs). X-Linked inhibitor of apoptosis protein (XIAP) is one of the best characterized IAPs. XIAP suppresses apoptosis by forming complexes with cysteine-aspartic proteases (caspase), through one of its baculovirus IAP repeat (BIR) domains. Its activity is endogenously antagonized by a second mitochondria derived activator of caspase (Smac). The anti-apoptotic behaviour of XIAP and the critical role it plays in the apoptotic program makes the Smac-XIAP interaction an important drug target. To this end, our laboratory is interested in synthesizing biologically related Smac mimetics which can induce apoptosis in a MDA-MB-231 cell line. Efforts have focused on (1) understanding BIR domain binding sites which allow for this interaction, and (2) the design and synthesis of molecules which are much more effective at inducing apoptosis compared to other well known analogues. Through the synthesis and evaluation of various divalent Smac mimetics we have been able to support the hypothesis that the likely binding site on XIAP is the BIR3 domain. As well, through the synthesis of a library of novel compounds, as described in the thesis, we have been able to assess the nature of the linker which joins the two tetrapeptide units. In our effort to understand which domains Smac binds with, various divalent analogues were synthesized containing MeAVPI-linker-IPVMeA (forward-reverse) and MeAVPI-linker-MeAVPI (forward-forward) sequence, which incorporated linkers with varying degrees of flexibility. We hypothesized that the forward-forward divalent mimetics would have decreased activity compared to the peptides synthesized in a forward-reverse fashion. Lastly, information gathered from structure activity relationship (SAR) studies have shown that substituting the lysine (P2) and isoleucine residues (P4) in the AVPI protein can create more potent inducers of apoptosis than its native AVPI sequence. As one of the most potent Smac mimetic that has been previously made known contains an alkyne bridge at P2 and a large hydrophobic moiety at P4, we hypothesized that similar Smac mimetics containing a propargyl glycine residue at P2 and a bulky hydrophobic moiety at P4 will be much more potent in inducing apoptosis.

Programmed cell death (apoptosis) is the most common mechanism of cell death in eukaryotes. The ability of cancer cells to evade and inhibit apoptosis has become a hallmark feature of cancer. This is accomplished through a family of proteins known as the inhibitor of apoptosis proteins (IAPs). X-Linked inhibitor of apoptosis protein (XIAP) is one of the best characterized IAPs. XIAP suppresses apoptosis by forming complexes with cysteine-aspartic proteases (caspase), through one of its baculovirus IAP repeat (BIR) domains. Its activity is endogenously antagonized by a second mitochondria derived activator of caspase (Smac). The anti-apoptotic behaviour of XIAP and the critical role it plays in the apoptotic program makes the Smac-XIAP interaction an important drug target. To this end, our laboratory is interested in synthesizing biologically related Smac mimetics which can induce apoptosis in a MDA-MB-231 cell line. Efforts have focused on (1) understanding BIR domain binding sites which allow for this interaction, and (2) the design and synthesis of molecules which are much more effective at inducing apoptosis compared to other well known analogues. Through the synthesis and evaluation of various divalent Smac mimetics we have been able to support the hypothesis that the likely binding site on XIAP is the BIR3 domain. As well, through the synthesis of a library of novel compounds, as described in the thesis, we have been able to assess the nature of the linker which joins the two tetrapeptide units. In our effort to understand which domains Smac binds with, various divalent analogues were synthesized containing MeAVPI-linker-IPVMeA (forward-reverse) and MeAVPI-linker-MeAVPI (forward-forward) sequence, which incorporated linkers with varying degrees of flexibility. We hypothesized that the forward-forward divalent mimetics would have decreased activity compared to the peptides synthesized in a forward-reverse fashion. Lastly, information gathered from structure activity relationship (SAR) studies have shown that substituting the lysine (P2) and isoleucine residues (P4) in the AVPI protein can create more potent inducers of apoptosis than its native AVPI sequence. As one of the most potent Smac mimetic that has been previously made known contains an alkyne bridge at P2 and a large hydrophobic moiety at P4, we hypothesized that similar Smac mimetics containing a propargyl glycine residue at P2 and a bulky hydrophobic moiety at P4 will be much more potent in inducing apoptosis.

Thesis (Ph. D.)--Cornell University, January, 2009. / Vita. Includes bibliographical references (leaves 104-114).

Programmed cell death (apoptosis) is the most common mechanism of cell death in eukaryotes. The ability of cancer cells to evade and inhibit apoptosis has become a hallmark feature of cancer. This is accomplished through a family of proteins known as the inhibitor of apoptosis proteins (IAPs). X-Linked inhibitor of apoptosis protein (XIAP) is one of the best characterized IAPs. XIAP suppresses apoptosis by forming complexes with cysteine-aspartic proteases (caspase), through one of its baculovirus IAP repeat (BIR) domains. Its activity is endogenously antagonized by a second mitochondria derived activator of caspase (Smac). The anti-apoptotic behaviour of XIAP and the critical role it plays in the apoptotic program makes the Smac-XIAP interaction an important drug target. To this end, our laboratory is interested in synthesizing biologically related Smac mimetics which can induce apoptosis in a MDA-MB-231 cell line. Efforts have focused on (1) understanding BIR domain binding sites which allow for this interaction, and (2) the design and synthesis of molecules which are much more effective at inducing apoptosis compared to other well known analogues. Through the synthesis and evaluation of various divalent Smac mimetics we have been able to support the hypothesis that the likely binding site on XIAP is the BIR3 domain. As well, through the synthesis of a library of novel compounds, as described in the thesis, we have been able to assess the nature of the linker which joins the two tetrapeptide units. In our effort to understand which domains Smac binds with, various divalent analogues were synthesized containing MeAVPI-linker-IPVMeA (forward-reverse) and MeAVPI-linker-MeAVPI (forward-forward) sequence, which incorporated linkers with varying degrees of flexibility. We hypothesized that the forward-forward divalent mimetics would have decreased activity compared to the peptides synthesized in a forward-reverse fashion. Lastly, information gathered from structure activity relationship (SAR) studies have shown that substituting the lysine (P2) and isoleucine residues (P4) in the AVPI protein can create more potent inducers of apoptosis than its native AVPI sequence. As one of the most potent Smac mimetic that has been previously made known contains an alkyne bridge at P2 and a large hydrophobic moiety at P4, we hypothesized that similar Smac mimetics containing a propargyl glycine residue at P2 and a bulky hydrophobic moiety at P4 will be much more potent in inducing apoptosis.

The dissemination of tumor cells to distant organs i.e. metastasis is an exceedingly complex process leading to 90% of all cancer deaths. Despite being so clinically important, little is known about this process that requires tumor cells to leave the primary tumor site, intravasate and transport through the blood stream, extravasate and colonize at secondary sites leading to distant metastases. Survivin, a member of the IAP (Inhibitor of Apoptosis) family with known functions in apoptosis and mitosis, is highly expressed in aggressive tumors and is associated with poor prognosis and adverse clinical outcome. But the mechanistic role of survivin in metastatic dissemination has not been investigated. In this study, we demonstrate an important and novel role of survivin in activating a broad gene expression program in tumor cells. Of particular importance is the upregulation of a distinct class of cell adhesion molecules, particularly fibronectin. This IAP mediated gene regulation requires synergistic intermolecular cooperation between survivin and its related cofactor molecule, XIAP that results in activation of NF-κB dependent fibronectin gene expression. The binding of fibronectin with its cognate cell surface receptors initiates outside–in signaling leading to the autocrine and paracrine activation of cell motility kinases, FAK and Src, in turn leading to enhanced tumor invasion and metastasis. The importance of survivin and XIAP in the process of metastasis has also been demonstrated in vivousing intrasplenic injections in mouse models. Overall this study is the first to place survivin upstream of transcriptional activation of gene expression particularly fibronectin. In addition, it also demonstrates the importance of survivin-XIAP complex in mediating NF-κB activation which in turn switches on the expression of various target genes involved in tumor metastasis. Hence this study dissects the upstream and downstream requirements of survivin- XIAP complex mediated tumor dissemination and metastasis. Significance of this Study The hallmark of end-stage cancer is metastasis, an incurable condition almost invariably associated with death from disease. Despite a better understanding of the metastatic process, and the identification of key gene expression requirements of this pathway, the development of anti-metastatic therapies has lagged behind, with no viable options being currently offered in the clinical setting. Our findings that Inhibitor of Apoptosis (IAP) proteins functions as metastasis-promoting genes independently of cell survival, but through activation of cell motility could have important ramifications for the broader application of IAP antagonists currently in early clinical trials, as novel anti-metastatic therapies.