Thesis (M.S.)--West Virginia University, 1999. / Title from document title page. Document formatted into pages; contains vii, 93 p. : ill. (some col.) Includes abstract. Includes bibliographical references (p. 72-80).

Thesis (M.Sc. Materials and Process Engineering)--University of Waikato, 2006. / Title from PDF cover (viewed April 23, 2009) Includes bibliographical references (p. 100-104)

Thesis supervisor: Madeline A. Shea. Includes bibliographical references (p. 195-206).

In summary, this thesis has explored the anti-cancer mechanisms of novel medicinal compounds via targeting calpains or S100A4 in the treatment of colon cancer, which could facilitate future establishment of effective medicinal compounds in the treatment of metastatic colon cancers with known molecular targets.;The incidence of colon cancer in Hong Kong and worldwide is on a rising trend, while its metastatic development is the leading cause of cancer-related deaths. Understanding the molecular mechanisms of how tumors progress and metastasize to secondary sites, at both biological and genetic levels, could enable us to identify potential molecular targets in drug development. In the present study, we explored how manipulation of signaling pathways by targeting calpains and S100A4 could facilitate the development of anti-tumor and anti-metastatic drugs.;The study investigating drug targeting on S100A4 in both in vitro and in vivo models had shown that the pharmacological store-operated calcium channel blocker would suppress S100A4-mediated migration by weakening extracellular S100A4-mediated calcium responses. The effects on S100A4-induced metastasis formation were confirmed in vivo with reduced splenic tumor volume and decreased number of liver metastases. These results have provided new insights to correlate between S100A4 and calcium signaling, making an important step forward in characterizing the dependence of calcium homeostasis in the process of metastasis, providing a novel strategy for S100A4-mediated metastasis.;With respect to the targeting on calpains, it was discovered that total Astragalus saponins (AST) and cryptotanshinone (CPT) are effective anti-cancer agents that elicit the endoplasmic reticulum (ER) stress response. They act by upregulating the expression of glucose-regulated protein (GRP) 78, leading to the initiation of apoptosis when the ER recovery process begins to fail. In particular, CPT caused rapid and sustained increase in cytosolic calcium in colon cancer cells that was accompanied by early GRP78 overexpression. The increase in cytosolic calcium was blocked by pre-treatment of BAPTA-AM through depletion of the ER calcium store. In consistent with these, we also confirmed that CPT significantly increased calpain activity, which could be blocked by calcium chelator or calpain inhibitors. Furthermore, a dynamic interaction between GRP78 and calpain under ER stress was unveiled during AST or CPT exposure. The degree of association was increased following prolonged ER stress, and suppressed either as the ER recovery process failed or with the presence of calpain inhibitors. Besides, inhibition of calpain activity suppressed NF-κB activation (a consequence of ER stress) and substantially enhanced the effects of CPT to promote apoptosis. More importantly, it was confirmed that the effects of calpain inhibitors to sensitize colon cancer cells to ER stress-associated apoptosis are p53-dependent. The anti-tumorigenetic effects of CPT were further demonstrated in vivo in xenografted nude mice by trageting calpains and in combination with calpain inhibitors.

Cardiovascular disease and the associated endothelial dysfunction are characterized by leukocyte activation, decrease endothelial nitric oxide synthase (eNOS) activity, and increased endothelial cell adhesion molecules expression. This leads to the release of myeloperoxidase (MPO) by activated neutrophils and monocytes. MPO is a peroxidase enzyme that plays an important role in innate immune host defense, however it has been shown to play a pathogenic role in cardiovascular disease, mainly by causing endothelial dysfunction. The molecular mechanisms through which MPO induces endothelial damage are not fully understood. Calpains are a family of calcium-dependent proteases. Two calpain isoforms, µ- and m-calpain, are expressed in the vascular wall, including endothelial cells. Activation of calpains has been recently implicated in inflammatory disorders of the vasculature. The goal of this study was to test the hypothesis of a role for calpains in the molecular mechanism(s) through which MPO causes endothelial dysfunction and vascular inflammation. To explore if MPO activates calpain and to identify the calpain isoform(s) involved, we first studied the effects of MPO treatment on calpain activity in mouse lung microvascular endothelial cells (MMVEC). MMVECs were stimulated with 10 nmol/L MPO for 60, 120, 180, and 240 min. Using a fluorescent calpain activity assay, we found that MPO time dependently activates calpain in endothelial cells, with peak activity reached within 180 min. Using immunoblot analysis techniques we demonstrated that the calpain isoform targeted by MPO is µ-calpain. Interestingly, using a biotin switch assay,10 nmol/L MPO appears to activate the µ-calpain isoform via denitrosylation of its C-terminus domain. Using MMVECs, we studied the effects of the MPO/µ-calpain signaling on endothelial dysfunction. MMVECs were stimulated with 10 nmol/L MPO for 180 min. Expression levels of Protein Phosphatase 2 (PP2A), total 5' AMP-activated protein kinase (AMPK), Thr172 phospho-AMPK, total endothelial nitric oxide synthase (eNOS),Ser1177 phospho-eNOS, total protein kinase B (AKT), Ser473 phospho AKT, Adiponectin receptor 1 (AdipoR1), and Adiponectin receptor 2 (AdipoR2), were measured by immunoblot analysis. Interestingly, MPO impaired Thr172AMPK, Ser1177eNOS, but not Ser 473 AKT phosphorylation in a calpain dependent manner. On the other hand, MPO significantly increased the expression levels of PP2A. Inhibition of PP2A with okadiak acid decreased the phosphorylation levels of AMPK, and eNOS, indicating that PP2A is a downstream target of the MPO/calpain system. MPO treatment significantly increased the expression of vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells. Pharmacological inhibition of calpain activity attenuated expression of VCAM-1. MPO also decreased protein levels of AdipoR1, and AdipoR2 in a calpain dependent manner. The treatment of MMVEC with adiponectin in the presence of MPO was not able to restore AdipoR2 expression levels. Using genetically modified mice, we found evidence of reduced leukocyte adhesion to the aortic endothelium in response to MPO in µ-calpain deficient mice, compared to wild-type mice . These effects appears to be attributed to the endothelial calpain, since incubating wild type aortas with calpain deficient leukocytes did not reduce leukocyte adhesion to the endothelium. The data in this study first establish a role for calpain in the endothelial dysfunction and vascular inflammation of MPO, with MPO activating the µ-calpain isoform via denitrosylation. Our data also report that increased calpain activity downregulats the expression of a number of signaling molecules important for endothelial cell function. This study may provide the MPO/calpain/PP2A signaling pathway as a novel pharmacological targets for the treatment of inflammation-driven vascular disorders. / Biomedical Sciences

Graduation date: 2006 / We hypothesized that calpain activity is elevated in response to muscle damage. To test this hypothesis, we examined the degradation of α-fodrin into its 150 and 145 kDa fragments following either 20 eccentric or isometric contractions. In addition, experiments were performed in the presence or absence of E-64-d, a calpain inhibitor. Both EDL and SOL muscles displayed significant differences (p<0.003 and p<0.002 respectively) between the raw and normalized 150 and 145 kDa α-fodrin fragments of the DMSO + E-64-d compared to the other bath treatments. Based on our model of exercise-induced muscle damage, we expected to see greater levels of 150 and 145 kDa α-fodrin fragments in those muscles that performed the eccentric protocol. However, there was no evidence that eccentric muscle damage increased the levels of 150 and 145 kDa α-fodrin fragments over the levels observed in the isometric trials. These findings suggest that the magnitude of damage was insufficient to activate calpains.

Podosomes are adhesive and invasive structures which may play an important role in numerous physiological and pathological conditions including angiogenesis, atherosclerosis, and cancer metastasis. Recently, the cysteine protease m-calpain (m-Capn) has been shown to cleave cortactin, an integral component of the podosomal F-actin core, as well as various proteins found in the peripheral adhesive region leading to the disassembly of these dynamic structures. In this study, I investigated whether Capn plays a role in the formation of podosomes downstream of c-Src. I show that: 1) phorbol-12, 13-dibutyrate (PDBu) as well as c-Src-Y527F expression induces podosome formation in mouse embryonic fibroblasts; 2) PDBu- and constitutively active c-Src-induced podosome formation is inhibited by the knockout of the m- and µ-Capn small regulatory subunit Capn4 in mouse embryonic fibroblasts (Capn4-/-), but is partially restored by re-expression of Capn4; 3) Capn4 localizes to podosomes; and 4) Inhibition of m- and µ-Capn proteolytic activity by the cell permeable calpain inhibitors has little effect on the formation of podosomes downstream of active c-Src. I conclude that Capn4 may play a role in the assembly phase of podosomes independent of calpain proteolytic activity. Work done in collaboration to determine a possible mechanism of action for the role of Capn4 in podosome assembly indicates that a possible binding partner of Capn4, β-PIX, co-localizes with, and shows in vivo association with Capn4. Furthermore, β-PIX and Capn4 bind directly in vitro in the presence of Ca2+. We conclude that Capn4 plays a role in podosome assembly, and this role may be through direct interaction with β-PIX in a calcium-dependent manner. / Thesis (Master, Biochemistry) -- Queen's University, 2008-09-26 16:16:00.768

Shigella, l’agent de la dysenterie bacillaire, envahit la muqueuse colique, causant une inflammation intense et destruction tissulaire. Afin de promouvoir l’infection, Shigella injecte des facteurs de virulence par l’intermédiaire d’un appareil de sécrétion de type III (T3SS) dans les cellules hôte, qui permettent la réorganisation du cytosquelette d’actine, régulent l’inflammation et l’intégrité du tissu. Parmi ces facteurs injectés par la bactérie, la protéine IpgD agit comme une phosphatidylinositol (PI) (4,5) bisphosphate phosphatase déphosphorylant le PIP2 en PI(5)P. L’hydrolyse du PI(4,5)P2 favorise la polymérisation de l’actine durant l’invasion des cellules épithéliales et régule négativement la migration des lymphocytes T. Il a également été démontré que le PI(5)P produit par IpgD active la voie de survie cellulaire dépendant de la PI(3) kinase et de la kinase AKT. Mon projet de thèse a pour but de caractériser le rôle d’IpgD dans les contrôles des réponses calciques durant l’invasion des cellules épithéliales par Shigella. Nous avons montré qu’IpgD limite le recrutement de récepteurs à l’inositol-triphosphate (InsP3) au site d’invasion. Des expériences d’imagerie calcique indiquent que durant les étapes précoces de l’invasion, IpgD favorise l’émission de réponses locales aux sites d’invasion de Shigella et limite celle de réponses globales. Des expériences de recouvrement de fluorescence après photo-blanchiment (FRAP) indiquent que les foyers d’invasion induits par le mutant ipgD montrent une diffusion moins restreinte que celle observée dans les foyers induits par la souche sauvage. Des études de modélisation supportent la notion qu’en limitant la production locale d’InsP3 et sa diffusion aux sites d’invasion, IpgD participe au confinement des réponses calciques locales en empêchant l’émission de réponses globales. Nous avons montré que lors de cinétiques d’infection prolongée, IpgD inhibe les réponses calciques globales dépendant de l’InsP3 induites par Shigella ou par des agonistes. L’inhibition de ces réponses conduit à un retard de l’activation de la calpaine, de la dégradation de la taline, une protéine des adhérences focales, ainsi que de la mort des cellules durant la réplication bactérienne intracellulaire. Nos résultats indiquent qu’IpgD est un effecteur critique de Shigella permettant de réguler la transition des réponses calciques locales vers des réponses globales et préservant les cellules infectées de la mort liée à une perte d’adhérence. / Shigella, the agent of bacillary dysentery, invades colonic epithelial cells where it disseminates causing an intense inflammation and tissue destruction. To efficiently promote infection, Shigella injects virulence effectors via a type III secretion system (T3SS) into host cell to divert processes involved in cytoskeletal rearrangements and processes regulating inflammatory signals or tissue integrity. Among the Shigella T3SS effectors, IpgD acts as a phosphatidylinositol (PI) (4, 5) bisphosphate phosphatase, which dephosphorylates PI (4,5) P2 to generate PI(5)P. IpgD-mediated hydrolysis of PI (4, 5) P2 favors actin polymerization during cell invasion and negatively regulates the migration of T cells. PI(5)P produced by IpgD was also shown to up-regulate the PI3K/AKT cell survival pathway and recycling of the Epidermal Growth Factor receptor. My project thesis focuses on the role of IpgD in the control of calcium responses during Shigella infection. We show that that IpgD is responsible for a decrease in the recruitment of inositol-triphosphate (InsP3) receptors at invasion sites. Ca2+-imaging experiments indicate that during the early stages of bacterial invasion, IpgD favors the elicitation of local Ca2+responses at Shigella invasion sites, and limits the induction of global Ca2+ responses. Fliuorescence Recovery After Photobleaching (FRAP) experiments indicate that actin foci induced by the ipgD mutant show faster diffusion kinetics than those in foci induced by WT. Modeling studies support the notion that the local decrease in InsP3 levels and of its diffusion kinetics triggered by IpgD at entry sites accounted for the modulation of local to global Ca2+ responses during Shigella invasion. We show that over prolonged infection kinetics, IpgD inhibits InsP3-dependent global Ca2+ responses induced by Shigella or agonists. IpgD-mediated inhibition of Ca2+ signals delays the activation of calpain, the degradation of the focal adhesion protein talin, and cell death during bacterial intracellular replication. Our results provide evidence that IpgD is a critical T3SS effector of Shigella regulating the transition from local to global Ca2+ signals, and preserving cells from death linked to loss of adhesion.

Traumatic brain injury (TBI) is a devastating health problem based on its high incidence, economic burden, and lack of effective pharmacological treatment. Individuals who suffer an injury often experience lifelong disability. TBI results in abrupt, initial cell damage leading to delayed neuronal death. The calcium-activated proteases, calpains, are known to contribute to this secondary neurodegenerative cascade. Prolonged activation of calpains results in proteolysis of numerous cellular substrates including cytoskeletal components, membrane receptors, and cytosolic proteins, contributing to cell demise despite coincident expression of calpastatin, the specific inhibitor of calpains. A comprehensive analysis using two separate calpastatin transgenic mouse lines was performed to test the hypothesis that calpastatin overexpression will reduce posttraumatic calpain activity affording neuroprotection and behavioral efficacy. Increased calpastatin expression was achieved using transgenic mice that overexpress the human calpastatin (hCAST) construct under control of a neuron-specific calcium-calmodulin dependent kinase II alpha or a ubiquitous prion protein promoter. Both transgenic lines exhibited enhanced calpastatin expression within the brain, extending into peripheral tissues under the prion protein promoter. hCAST overexpression significantly reduced protease activity confirmed by reductions in acute calpain-mediated substrate proteolysis in the cortex and hippocampus following controlled cortical impact brain injury. Aspects of posttraumatic motor and cognitive behavioral deficits were also lessened in hCAST transgenic mice compared to their wildtype littermates. However, volumetric analyses of neocortical contusion revealed no histological neuroprotection at either acute or long-term time points in either transgenic line. Partial hippocampal neuroprotection observed at a moderate injury severity in neuron-specific calpastatin overexpressing transgenic mice was lost after severe TBI. Greater levels of calpastatin under the prion protein promoter line failed to protect against hippocampal cell loss after severe brain injury. This study underscores the effectiveness of calpastatin overexpression in reducing calpain-mediated proteolysis and behavioral impairment after TBI, supporting the therapeutic potential for calpain inhibition. However, the reduction in proteolysis without accompanied neocortical neuroprotection suggests the involvement of other factors that are critical for neuronal survival after contusion brain injury. Augmenting calpastatin levels may be an effective method for calpain inhibition and may have efficacy in reducing behavioral morbidity after TBI and neurodegenerative disorders.

Les cellules souche cancéreuses contribuent au développement des sarcomes, mais le manque de marqueurs spécifiques empêche leur caractérisation et la possibilité de cibler ce type de cellules. Nous avons utilisé la séquence régulatrice de la calpaïne-6 dans des systèmes rapporteurs pour identifier les cellules exprimant la calpaïne-6. Ces cellules étaient des cellules initiatrices de tumeurs et se comportaient comme des cellules souche, au sommet de la hiérarchie cellulaire. L'expression de la calpaïne-6 dépend d’un programme génique de cellules souche qui implique Oct4, Nanog et Sox2 et est activée par l'hypoxie. L’inhibition de la calpaïne-6 a bloqué le développement tumoral et a induit la diminution du nombre de cellules souche cancéreuses dans les sarcomes osseux. L’expression de la calpaïne-6 était inversement corrélée à l'expression de marqueurs de sénescence mais était associé à un flux autophagique dynamique. L’inhibition de la calpaïne-6 a induit l'entrée des cellules en sénescence et a supprimé le flux autophagique. Nos résultats révèlent que le calpaïne-6 identifie les cellules souche des sarcomes et joue un rôle important dans le maintien des cellules souche cancéreuses en contrôlant les processus d’autophagie et de sénescence. La calpaïne-6 semble être une cible thérapeutique prometteuse pour éradiquer les cellules souche dans les sarcomes / Cancer stem cells contribute to sarcoma development, but lack of specific markers prevents their characterization and the possibility of targeting. We used the regulatory sequence of calpain-6 in reporter constructions to identify calpain-6–expressing cells. These cells were tumor-initiating cells and behaved like stem cells at the apex of the cellular hierarchy. Calpain-6 expression depended on the stem-cell transcription network that involves Oct4, Nanog, and Sox2 and was activated by hypoxia. Calpain-6 knockdown blocked tumor development and induced depletion of sarcoma stem cells. Calpain-6 was inversely associated with expression of senescence markers but was associated with a dynamic autophagy flux. Calpain-6 knockdown induced cell entry into senescence and suppressed autophagy flux. Our results reveal that calpain-6 identifies sarcoma stem-cell and plays an important role as a regulator of cancer cell fate driving a switch between autophagy and senescence. Calpain-6 may be a promising therapeutic target to eradicate sarcoma stem cells.