Dissertations / Theses on the topic 'Sialyltransferases'

Sialic acids terminate oligosaccharide chains on the surfaces of mammalian cells and many microbial species, often playing critical biological roles in recognition and adherence. The enzymes which transfer the sialic acid moiety to these key terminal positions are known as sialyltransferases. Despite their important biological roles very little is understood about the mechanism of action or molecular structure of these enzymes. Campylobacter jejuni, a highly prevalent food-borne pathogen that causes acute gastroenteritis in humans, contains two versions of a sialyltransferase: a monofunctional α-2,3-sialyltransferase Cst-I and a bifunctional α-2,3/8-sialyltransferase Cst-II. Both of these enzymes are responsible for lipooligosaccharides (LOS) sialylation to camouflage the bacterial surface from the host, and thus evade the immune system. In addition, sialylated-glycoconjugates on C. jejuni often mimic human gangliosides, contributing to the molecular basis of Guillain-Barré syndrome, an autoimmune disease of the peripheral nervous system that often develops post-infection. The sialyltransferase reaction is believed to proceed through an inversion mechanism, catalyzing the transfer of sialic acid from CMP-N-acetylneuraminic acid onto different acceptors. This thesis is to understand through high-resolution structural characterization, site specific mutagenesis and kinetic analysis, the mechanism of the glycosyl transfer(s) in both monofunctional and bifunctional Csts. Crystals of Cst-II were obtained and the complex structures with bound CMP, inert donor sugar analogue CMP-3-fluoro-N-acetylneuraminic acid (CMP-3FNeu5Ac) and inhibitor CDP were solved using MAD phasing from incorporated selenomethionines. Work within this study represents the first known structure of a sialyltransferase. Based on the position of the substrates, the active site of Cst-II has been elucidated. Site-directed mutagenesis of conserved residues in the active site was performed and mutants were characterized using enzyme kinetics. A reaction mechanism was proposed based on the kinetic assay. A directed evolution methodology was designed for glycosyltransferases using Cst-II as the model system. A single mutation, F91Y was found to substantially increase the reaction rate of the enzyme with a fluorescent-coupled acceptor, bodipy-lactose. The crystal structure of this Cst-II F91Y mutant was solved and it revealed an unexpected flip of the tyrosine side chain of Y91 from the core of the enzyme into the solvent region, exposing a hydrophobic pocket which seems to be capable of accommodating the bodipy ring structure. Together with kinetic analyses, the crystallographic study was able to explain the observed increase in the catalytic rate for this novel sugar acceptor. A monofunctional variant of Cst, Cst-I, also isolated from Campylobacter jejuni, was characterized crystallographically and kinetically. The conservation of active site residues supports the proposed mechanism for GT-42 sialyltransferases. The complex structure of the Cst-I enzyme with the donor analogue CMP-3FNeu5Ac provides a platform for molecular modeling of various acceptors into the active sites of Cst-I and Cst-II. The modeling shed lights upon the understanding of differences in substrate specificity. The structures of these complexes will be used as templates to design therapeutic inhibitors against this common human pathogen.
Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
Graduate

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CAPES, CNPQ, FACEPE
O câncer de mama feminino é o segundo tipo de câncer mais frequente no mundo, com aumento de incidência de 22% a cada ano. Estudos nas últimas décadas revelaram que a transformação maligna está associada a uma variedade de células com padrões de glicosilação alterados, como por exemplo a sialilação. Os ácidos siálicos tem sido relacionados à iniciação e progressão do câncer, tendo assim implicações potenciais na prevenção, diagnóstico e tratamento da doença. Este trabalho avaliou a expressão fenotípica de ST3Gal-I, através da imunohistoquímica, e o perfil de ácido siálico, através da histoquímica com lectinas usando Maackia Amurensis agglutinin II (MAA), em tecidos mamários diagnosticados com fibroadenoma (n=59), carcinoma ductal in situ (CDIS, n=40), carcinoma ductal invasivo (CDI, n=50) e carcinoma lobular (CL, n=42). Todos os tipos de lesões de mama apresentaram alta imunopositividade à ST3Gal-I, sendo observada uma expressão em 93,2% dos casos de Fibroadenoma, 92,5% de CDIS, 96% dos casos CDI e 85,2% de CL. As células apresentaram um padrão de marcação citoplasmático e perinuclear com relação à ST3Gal-I. Diferentes distribuições de resíduos de ácido siálico ⍺2,3-ligados, com um padrão de marcação predominantemente citoplasmático e membranar, foram observados nas lesões de mama estudadas. Os casos de fibroadenoma apresentaram o menor percentual de sialilação entre as lesões analisadas (47,5%), enquanto os de CDI o maior pecentual (98%). Embora este estudo não tenha mostrado diferença significativa na expressão de ST3Gal-I entre as lesões de mama, alterações representativas na presença de ácidos siálicos entre fibroadenoma e lesões malignas (p<0,0001), e também entre CDIS e CDI (p = 0.037) foram notadas. Não foram encontradas correlações significativas entre as expressões de ST3Gal-I e MAA, os marcadores de rotina e as características clinico-histopatológicas dos pacientes. Os resultados indicam uma distribuição particular de ácidos siálicos ⍺2,3-ligados nas células mamárias entre as lesões estudadas sugerindo seu envolvimento na progressão/manutenção do câncer de mama.
The female breast cancer is the second most common type of cancer in the world, with increased incidence of 22% every year. Recent studies in recent decades have revealed that the malignant transformation is associated with a variety of cells with altered glycosylation patterns such as sialylation. Sialic acids have been demonstrated to participate in cancer initiation and progression, thus has potential implications for cancer prevention, diagnosis and treatment. This study evaluated the phenotype expression of ST3Gal-I using immunohistochemistry and the sialic acid residues profile by histochemistry with Maackia amurensis agglutinin II (MAA) in mammary tissues diagnosed as fibroadenoma (n=59), ductal carcinoma in situ (DCIS, n=40), invasive ductal carcinoma (IDC, n=50) and lobular carcinoma (LC, n=42). All types of breast lesions showed high ST3Gal-I immunopositivity, its expression was observed in 93.2% cases of Fibroadenoma, 92.5% of DCIS, 96% of IDC and 85.2% cases of LC. The cells ST3Gal-I staining pattern was mainly cytoplasmatic and perinuclear. The MAA staining in breast lesions showed a diffuse cytoplasmatic and membrane pattern with different distribution of ⍺2,3-linked sialic acids among the lesions studied, fibroadenoma cases showed the lowest percentage among the analyzed lesions (47.5%) while IDC showed the highest (98%). Although this study did not show a significant difference in expression of ST3Gal-I among all lesions, representative alterations in sialic acid content between fibroadenoma and malignant lesions (p<0.0001), and also between CDIS and CDI (p=0.037) were observed. No significant correlations were found between the expressions of ST3Gal-I and MAA, routine markers and clinical-histopathological characteristics of the patients. Results indicate different distribution of ⍺2,3-linked sialic acids on the cells of the studied lesions which seems to be involved in breast cancer progression and/or maintenance.

Este trabajo demuestra que los genes que codifican para los enzimas beta-galactosido alfa-2,3-sialiltransferasa 3 (ST3Gal III), y en menor medida beta-galactosido alfa-2,3-sialiltransferasa 4 (ST3Gal IV), están directamente implicados en etapas clave de la progresión tumoral como la adhesión, la migración y la formación de metástasis en las líneas de adenocarcinoma pancreático humano Capan-1 y MDAPanc-28. También, que las Especies Reactivas del Oxígeno (ROS) generadas durante los procesos de proliferación y diferenciación celular o debido a estímulos oxidantes externos, desempeñan un importante papel en el control de la síntesis de ST3Gal III y SLex, y por lo tanto en la regulación del fenotipo metastático. Además, junto al papel pro-adhesivo de la E-Selectina, este trabajo ha descrito efectos prometastáticos adicionales para esta molécula como inductora de la migración y de la secreción de VEGF a través de un mecanismo E-Selectina-SLex dependiente.
This work shows that genes that codifying for the enzymes beta-galactoside alpha-2,3-sialyltransferase 3 (ST3Gal III), and in a lower extent beta-galactoside alpha-2,3-sialyltransferase 4 (ST3Gal IV) , are directly implicated in key steps of tumour progression such as adhesion, migration and metastasis formation in the pancreatic adenocarcinoma cell lines Capan-1 and MDAPanc-28. Moreover, Reactive Oxygen Species (ROS) generated in these cell lines during cell proliferation-differentiation processes or by external oxidant stimuli, play a role in the control of ST3Gal III and SLex levels and in the acquisition of a more aggressive phenotype. And, together with the pro-adhesive role of E-Selectin for circulating cells, this work uncovers sE-Selectin dependent migration and VEGF secretion through a SLex depending mechanism, supporting additional pro-metastatic effects for sE-Selectin-SLex interaction.

Sialyltransferases, CMP-sialic acid synthetases and CMP-sialic acid transport proteins play a crucial role in the construction of cell surface glycoconjugates. These proteins also have a pivotal role to play in a number of diseases, including cancer. The sialyltransferase enzymes are responsible for transfering sialic acids from the donor substrate (CMP-sialic acid) to growing cell surface glycoconjugate chains within the Golgi apparatus. The CMP-sialic acid synthetase enzymes are responsible for the synthesis of the CMP-sialic acid, the donor substrate of the sialyltransferases in the nucleus, while the CMP-sialic acid transport proteins are responsible for transporting CMP-sialic acid from the Cytosol to the Golgi apparatus. When these proteins function in an abnormal way, hypersialylation results, leading to an increased level of sialylation on the cell surface. This increased level of sialylation aids in the detachment of primary tumour cells due to an increase in the level of overall negative charge, causing repulsion between the cancer cells. Therefore, the sialyltransferase enzymes, CMP-sialic acid synthetases and CMP-sialic acid transport proteins are intimately involved in the metastatic cascade associated with cancer. Chapter 1 provides a general introduction of cancer metastasis, discussing the roles of three target proteins (CMP-sialic acid synthetases, CMP-sialic acid transport proteins and sialyltransferases), as well as discussing their substrate specificities, with an emphasis on their involvements in cancer metastasis. The Chapter concludes with an overview of the types of compounds intended to be utilised as probes or inhibitors of these proteins. Chapter 2 describes the general approach towards the synthesis of CMP-Neu5Ac mimetics with a sulfur linkage in the presence of a phosphate group in the general structure 38. The precursor phosphoramidite derivative 45 was prepared and isolated in a good yield using Py.TFA. Unfortunately, the target compound 38 could not be prepared. Chapter 3 describes an alternative strategy wherein S-linked sialylnucleoside mimetics, of the general structure 39, with a sulfur linkage, but no phosphate group, between the sialylmimetic and the ribose moiety in the base is targeted. A series of these S-linked sialylnucleoside mimetics were successfully prepared. Cytidine, uridine, adenosine and 5-fluorouridine nucleosides were used to create a library of different nucleosides and with structural variability also present in the sialylmimetic portion. This small 'library' of 15 compounds was designed to shed light on the interaction of these compounds with the binding sites of the sialyltranferase, CMP-sialic acid synthetase and/or CM-sialic acid transport protein. Approaches towards the synthesis of O-linked sialylnucleoside mimetics of the general structure 40 are described in Chapter 4. Several methodologies are reported, as well as protecting group manipulations, for successful preparation of these sialylnucleoside mimetics. Cytidine and uridine were employed as the nucleosides, thus allowing a direct comparison between the O- and S-linked sialylnucleoside mimetics in biological evaluation. It appears from these synthetic investigations that gaining access into the O-linked series is not as straightforward as for the S-linked series, with alternative protecting group strategies required for the different nucleosides. The biological evaluation of some of the compounds reported in Chapters 3 and 4 is detailed in Chapter 5. The sialylnucleoside mimetics were evaluated, by 1H NMR spectroscopy, for their ability to inhibit CMP-KDN synthetase. In addition, an initial 1H NMR spectroscopic-based assay was investigated for inhibition studies of α(2,6)sialyltranferase in the absence of potential inhibitors. The final chapter (Chapter 6) brings together full experimental details in support of the compounds described in the preceding Chapters.

Sialyltransferases, CMP-sialic acid synthetases and CMP-sialic acid transport proteins play a crucial role in the construction of cell surface glycoconjugates. These proteins also have a pivotal role to play in a number of diseases, including cancer. The sialyltransferase enzymes are responsible for transfering sialic acids from the donor substrate (CMP-sialic acid) to growing cell surface glycoconjugate chains within the Golgi apparatus. The CMP-sialic acid synthetase enzymes are responsible for the synthesis of the CMP-sialic acid, the donor substrate of the sialyltransferases in the nucleus, while the CMP-sialic acid transport proteins are responsible for transporting CMP-sialic acid from the Cytosol to the Golgi apparatus. When these proteins function in an abnormal way, hypersialylation results, leading to an increased level of sialylation on the cell surface. This increased level of sialylation aids in the detachment of primary tumour cells due to an increase in the level of overall negative charge, causing repulsion between the cancer cells. Therefore, the sialyltransferase enzymes, CMP-sialic acid synthetases and CMP-sialic acid transport proteins are intimately involved in the metastatic cascade associated with cancer. Chapter 1 provides a general introduction of cancer metastasis, discussing the roles of three target proteins (CMP-sialic acid synthetases, CMP-sialic acid transport proteins and sialyltransferases), as well as discussing their substrate specificities, with an emphasis on their involvements in cancer metastasis. The Chapter concludes with an overview of the types of compounds intended to be utilised as probes or inhibitors of these proteins. Chapter 2 describes the general approach towards the synthesis of CMP-Neu5Ac mimetics with a sulfur linkage in the presence of a phosphate group in the general structure 38. The precursor phosphoramidite derivative 45 was prepared and isolated in a good yield using Py.TFA. Unfortunately, the target compound 38 could not be prepared. Chapter 3 describes an alternative strategy wherein S-linked sialylnucleoside mimetics, of the general structure 39, with a sulfur linkage, but no phosphate group, between the sialylmimetic and the ribose moiety in the base is targeted. A series of these S-linked sialylnucleoside mimetics were successfully prepared. Cytidine, uridine, adenosine and 5-fluorouridine nucleosides were used to create a library of different nucleosides and with structural variability also present in the sialylmimetic portion. This small 'library' of 15 compounds was designed to shed light on the interaction of these compounds with the binding sites of the sialyltranferase, CMP-sialic acid synthetase and/or CM-sialic acid transport protein. Approaches towards the synthesis of O-linked sialylnucleoside mimetics of the general structure 40 are described in Chapter 4. Several methodologies are reported, as well as protecting group manipulations, for successful preparation of these sialylnucleoside mimetics. Cytidine and uridine were employed as the nucleosides, thus allowing a direct comparison between the O- and S-linked sialylnucleoside mimetics in biological evaluation. It appears from these synthetic investigations that gaining access into the O-linked series is not as straightforward as for the S-linked series, with alternative protecting group strategies required for the different nucleosides. The biological evaluation of some of the compounds reported in Chapters 3 and 4 is detailed in Chapter 5. The sialylnucleoside mimetics were evaluated, by 1H NMR spectroscopy, for their ability to inhibit CMP-KDN synthetase. In addition, an initial 1H NMR spectroscopic-based assay was investigated for inhibition studies of ?(2,6)sialyltranferase in the absence of potential inhibitors. The final chapter (Chapter 6) brings together full experimental details in support of the compounds described in the preceding Chapters.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Institute for Glycomics
Full Text

La myogenèse est un processus physiologique qui inclut la prolifération, la différenciation et la fusion des myoblastes dans le but de créer des myotubes et par conséquent des fibres musculaires. Ces processus sont régulés par les MRF (Myogenic Regulatory Factors) et par les protéines Pax3 et Pax7, membres de la famille des protéines « Paired box ». L'équilibre entre l'expression de Pax7 et de MyoD est le facteur déterminant du devenir des progéniteurs musculaires et de leur entrée en différenciation. La fusion myoblastique nécessitent aussi la présence des glycanes sur les protéines à la surface cellulaire. Nous avons démontré qu'au cours de la différenciation myoblastique a lieu une diminution de l'α2,6 sialylation associée à une baisse de l'expression du gène st6gal1. Nous avons utilisé la lignée myoblastique murine C2C12 afin d'analyser les modifications de l'α2,6 sialylation et son impact sur l'expression de Pax7 au cours de la différenciation cellulaire. C'est pourquoi, nous avons créé des clones de C2C12 qui sous-expriment st6gal1 grâce à l'introduction d'un shRNA spécifique. Les clones obtenus montrent une prolifération ralentie et une faible expression de Pax7 ainsi qu'un indice de fusion plus important. Les structures glycaniques modifiées lors de la différenciation ont été identifiées par « lectinblots » et spectrométrie de masse : les N-glycanes sialylés bi-fucosylés sont les plus impactés par la diminution de l'α2,6 sialylation en différenciation. L'indice de fusion observé dans les clones engendre une diminution de la proportion des cellules de réserve (Pax7+/MyoD-). La quasi-extinction de l'expression de Pax7 pendant la différenciation est due à la baisse de l'activation de la voie de Notch. Le KD de st6gal1 inactive la voie Smad2/3 associée à la prolifération et il active la voie Akt connue pour favoriser la différenciation. Ainsi, nous montrons que l'α2,6 sialylation est nécessaire pour la prolifération myoblastique et elle contribue à l'activation de Pax7
Myogenesis is a physiological process which includes the proliferation, the differentiation and the fusion of myoblasts in order to form myotubes and then muscular fibers. These mechanisms are regulated by the MRF (Myogenic Regulatory Factors) and Pax3 and Pax7 proteins which are members of the Pax protein family. The balance between Pax7 and MyoD is the determining factor of the cell fate determination and the entry in differentiation. The myoblasitc fusion requires the presence of glycans on cell surface proteins. We demonstrated that during C2C12 cell differentiation, takes place a decrease of α2,6 sialylation associated with a decrease of gene expression coding for ST6Gal I. We have used the C2C12 myoblastic cell line to analyse the modifications of α2,6 sialylation and its impact on the expression of Pax7 during cell differentiation. For this purpose, we have created C2C12 clones which expressed shRNA against st6gal1. These cells show a slowdown proliferation, a severe decrease of Pax7 expression and an earlier entry in differentiation. The glycanic structures modified during myoblast differentiation were identified by lectinoblotting and mass spectrometry: the sialylated bi-fucosylated N-glycans are the most impacted by the decrease of α2,6 sialylation. The earlier fusion of the clones st6gal1- leads to a decrease of the amount of the reserve cells (Pax7+/MyoD-). The extinction of Pax7 expression during differentiation is due to the decrease of the activation of Notch pathway. The KD of st6gal1 inactivates Smad2/3 pathway associated with the cell proliferation and it activates the Akt pathway which favors the cell fusion. We can conclude that α2,6 sialylation is required for C2C12 proliferation and it contributes to Pax7 activation

Chez les mammifères, les acides sialiques se trouvent aux extrémités terminales des glycoconjugués (glycoprotéines et glycolipides) ou des oligosaccharides libres. De part cette position terminale sur les glycannes des glycoconjugués et de leur charge négative, les acides sialiques conditionnent les propriétés physico-chimiques des sialoglycoconjugués et sont souvent impliqués dans des mécanismes d'interaction cellulaire. L'association de résidus d'acides sialiques par des liaisons en α2,8 forme des chaînes linéaires de longueur variable classées selon le degré de polymérisation en structures disialylées (diSia), oligosialylées (oligoSia) ou polysialylées (polySia, ou PSA pour Poly Sialic Acid). Les PSA portés par la N-CAM (Neural Cell Adhesion Molecule) ont été les premiers mis en évidence chez les mammifères. Ils sont impliqués dans de nombreux processus physiologiques tels que la migration des cellules neuronales, la croissance axonale ou la synaptogenèse. Depuis, d'autres structures di-, oligo- et polysialylées ont été décrites chez les mammifères et chez l'homme en particulier, mais très peu d'études concernant leurs implications biologiques et leur biosynthèse ont été menées. Grâce à une analyse in silico, nous avons reconstitué un gène putatif d'α2,8-sialyltransférase sur le chromosome 10 humain, que nous avons nommé hST8Sia VI. Les analyses de l' expression de ce gène dans différents tissus et cellules humains ont mis en évidence une expression faible et ubiquiste de ce gène. Nous avons cloné la totalité du cadre ouvert de lecture de ce gène à partir des cellules cancéreuses mammaires MCF-7 et avons produit une forme recombinante soluble de hST8Sia VI dans des cellules COS-7 et Sf-9 pour caractériser son activité enzymatique. Nos tests d'activité in vitro ont démontré que hST8Sia VI catalyse le transfert d'un seul résidu d'acide sialique sur les motifs glucidiques sialylés en α2-3 trouvés sur les O-glycannes des glycoprotéines pour former des motifs disialylés du type Neu5Acα2-8Neu5Acα23Galβ 1-3GalNAc-. Afin de déterminer son activité enzymatique in vivo, nous avons créé un modèle de cellules cancéreuses mammaires (MDA) sur-exprimant stablement hST8Sia VI. Nos premiers résultats indiquent que hST8Sia VI utilise préférentiellement les motifs α2,3-sialylés, ce qui confirme les analyses réalisées in vitro.

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O câncer de pele ou tumor cutâneo não-melanoma (TCNM), é a neoplasia maligna de maior incidência no Brasil. Os cânceres, em geral, apresentam padrão de glicosilação aberrante na estrutura de oligossacarídeos de superfície celular (fenótipo), determinada pela ação conjunta de glicosiltransferases (genótipo). O presente estudo objetiva avaliar o perfil glicofenotípico de glicoconjugados de superfície celular de TCNM e correlacionar com o padrão de expressão de sialiltransferases. Cortes (4μm) de biópsias de Carcinoma Basocelular (CBC), Carcinoma Espinocelular (CEC), Ceratose Actínica (CA) e Ceratoacantoma (KA) foram ensaiados com as lectinas SNA, Sambucus nigra agglutinin, (específica para ácidos siálicos ligados na posição α-2,6) e MAA, Maakia amurensis agglutinin (específica para ácidos siálicos na posição α-2,3). A inibição da ligação lectina-carboidrato foi realizada com o ácido siálico (300mM). As amostras também foram avaliadas com anticorpos monoclonais anti-ST3Gal I (específico para β-galactosideo α2,3 sialiltransferase) e anti- ST6Gal I (específico para β-galactosideo α2,6 sialiltransferase). Semelhanças no padrão de sialilação entre CEC, CA e KA foram encontradas na histoquímica com lectinas e, principalmente na imunohistoquímica. O CBC não apresentou marcação na maioria dos casos para ambas as técnicas, indicando que este tipo tumoral apresenta um padrão molecular próprio podendo estar associado ao seu comportamento biológico (baixo potencial invasivo e metastático). Os resultados indicam a importância da sialilação no desenvolvimento e manutenção de tumores cutâneos, sendo a histoquímica com lectinas e a imunohistoquímica ferramentas úteis na determinação deste tipo de glicosilação na superfície de células tumorais.

Sialic acids are involved in many biological processes. In glycoproteins and glycolipids they are essential for signalling and mediate molecular interactions as well as being targets for many pathogens such as influenza virus. The synthesis of sialylated glycoconjugates is of great importance. The incorporation of sialic acid through chemical synthesis carries several difficulties, enzymatic strategies using glycosyltransferases are very attractive alternative strategy, and have been used on a broad range of substrates forming glycosidic linkages with regio-and stereo-specificity. The work presented herein shows the study and application of two enzymes, Photobacteriumdamselae alpha2,6-sialyltransferase (Pd2,6ST) and Trypanosoma cruzi trans-sialidase (TcTS) which are used in the synthesis of sialyloligosaccharides. Both enzymes were expressed in E.coli and purified for biotransformations. In the first application new sialylated chromogenic compounds were generated through this enzymatically by using TcTS and a Pd2,6ST. These compounds were used for the detection of neuraminidase activity in a number of biological samples and led to the discovery of neuraminidase activity from Bacillus pumilus and Arthrobacter aurescens, two different bacteria in which the presence of neuraminidases had never been described. Secondly, TcTS was used to study lipid glycosylations. Glycans in biological systems can be associated to complex lipidic microdomains and the presence of these microdomains can affect the activity of some enzymes. In case of Trypanosoma cruzi trans-sialidase, a decreased activity was detected when the acceptor substrate was part of the aggregated lipid rafts compared to activity observed when the reaction was performed using fully dispersed substrate. Thirdly, the sialylation of glycoarrays using Pd2,6ST was studied. For the first time, sialylated glycans with alpha2,6- glycosidic linkages were successfully incorporated into a gold glycoarray platform, which had been previously developed for the label-free detection of carbohydrate-protein interactions. Successful enzymatic incorporation of sialic acids onto the arrays was confirmed with commercial available lectins. Finally, by using the gold glycoarray platform containing both 2,3 and 2,6 linked sialic acids as well as other common glycans, the carbohydrate-binding properties of the surface proteins of the bacterium Lactobacillus reuteri was studied using MALDI-ToF MS techniques. For first time, strong interactions were observed between a mucus binding protein and Neu5Ac alpha2,6-linked glycans, with much weaker binding to 2,3-linked analogues. Such glycan structures have been identified in abundant manner in colon mucins and this study contributes to the understanding of complex interactions between mucins and probiotic organisms as well as pathogenic bacteria. These studies show that glycan arrays can contribute both to the understanding of probiotics as well as to the identification of glycan binding proteins as targets for new drugs.

Serum levels of several molecules associated to pancreatic adenocarcinoma (PDAC) pathophysiology are evaluated in this work, in order to determine their diagnostic value, distinguishing between PDAC patients and healthy controls (HC), different gastrointestinal tumours (GIT) and chronic pancreatitis (CP). Plasma mRNA levels in plasma of sialyltransferases (ST3Gal III and ST3Gal IV) could differentiate between HC and PDAC. Moreover, lower levels of ST3Gal III in early stages of PDAC compared to PDAC advanced stages were reported. The Glasgow Prognostic Score (GPS), inflammatory response quantification, differentiated between all the study groups. IGF-1 levels were lower in neoplasic groups of patient vs HC and CP. We assessed the diagnostic capacity of different markers alone or in combination and compared with that of CA 19.9. The best diagnostic capacity was found combining CEA, CA 19.9 and IGF-1 compared with CA 19.9 and it could be useful to distinguish PDAC from CP.
En este trabajo se analizan distintos parámetros séricos relacionados con la fisiopatología del adenocarcinoma pancreático (PDAC), para evaluar su uso como marcadores que permitan distinguir entre pacientes con PDAC y controles sanos (C), otras neoplasias del tracto gastrointestinal (ONEOS) y pancreatitis crónica (PC). La expresión en plasma de sialiltransferasas, ST3Gal III y ST3Gal IV diferencia entre controles y PDAC. Así mismo, los niveles de ST3Gal III permiten diferenciar en PDAC entre estadíos iniciales y metastáticos. El Glasgow Prognostic Score (GPS), medida de la respuesta inflamatoria, diferencia entre todos los grupos del estudio. Los valores de IGF-1 disminuyen en procesos neoplásicos vs C y PC.Se analiza la capacidad diagnóstica de los distintos parámetros individualmente y combinados entre sí respecto al CA19.9. La combinación de CA 19.9, CEA, IGF-1 aumenta la precisión diagnóstica frente al CA19.9 y podría ser útil para distinguir PDAC de PC.

The mucus associated molecules intelectin (ITLN), resistin like molecule beta (RELMβ) and beta galactoside alpha 2-3 sialyltransferase (SIAT4C) are upregulated in nematode infections known to induce a typical T helper cell type 2 (Th2) response in mice. It was hypothesised that these three mucus-associated molecules were co-regulated by Th2 cytokines and that their upregulation was part of a typical anti-parasite response in other species. Sheep were chosen as a model because of the economic importance of both respiratory and gastrointestinal tract parasitic infections. Culture of a human colonic carcinoma cell line with either interleukin 4 (IL-4) or IL-13 confirmed the upregulation of ITLN and RELMβ in a Th2 environment however failed to show co-regulation with SIAT4C. Of the Th1 or Th2 cytokines examined only IFNγ had a significant effect on expression of SIAT4C transcript. ITLN transcript and protein was demonstrated in sheep tissue and furthermore three different ITLNs (sITLN1, sITLN2, sITLN3) which had a differential tissue distribution were cloned and sequenced. SIAT4C was widely expressed in sheep tissues and the full sequence was deduced. There was no evidence of expression of RELMβ sITLN transcripts were upregulated in response to IL-4 in an ex-vivo sheep tracheal explant culture model whilst sheep (s) SIAT4C was significantly downregulated in the same model. In a sheep model of infection with the abomasal nematode, Teladorsagia circumcincta, known to induce a Th2 biased response, sITLN transcripts and protein and sSIAT4C transcript were upregulated in response to a challenge infection. sITLN1 and sITLN2 were shown to upregulate at an earlier time point post challenge in previously infected (immune) compared to naïve yearling sheep and lambs and significant upregulation of sSIAT4C transcript was seen in challenged previously infected but not challenged naïve sheep and lambs. sITLNs and sSIAT4C may have an important role in the mucosal immune response to parasitic infections in sheep.

Au cours de ce travail, nous avons developpe une synthese chimio-enzymatique du synthon neu5ac-alpha-(2,3)-gal, present au niveau de nombreuses molecules d'interet biologique. L'etape cle de cette synthese est la condensation enzymatique de l'acide n-acetyl-neuraminique sur une unite galactosyl via l'alpha-(2,3)sialyltransferase de foie de porc. Nous avons tout d'abord recherche des analogues simplifies du substrat accepteur naturel, l'agt, relativement peu disponible pour des syntheses a grande echelle. Cette etude de specificite de l'enzyme nous a permis de trouver un analogue, le disaccharide gal-beta-(1,3)gal-2oac-ome, entrainant une activite relative de 100% par rapport au substrat naturel. Nous avons ensuite realise le couplage enzymatique pour obtenir le trisaccharide correspondant neu5ac-alpha-(2,3)gal-beta-(1,3)gal-(2oac)-ome. Ce trisaccharide conduit directement au synthon souhaite neu5ac-alpha-(2,3)gal, en subissant une reaction d'acetolyse partielle. Il est egalement un excellent substrat donneur d'acide sialique pour des reactions de trans-sialidation, via la trans-sialidase de trypanosoma cruzi. Ce travail est un bon exemple de l'utilisation de reactions de biotransformation dans des protocoles de synthese essentiellement chimiques

博士
國立陽明大學
臨床醫學研究所
90
Sialic acids including a number of their derivatives are ubiquitous at the terminal positions of oligosaccharides of glycoproteins. Due to their acidic nature, they impart a net negative charge to the cell surface and are important in cell-cell or cell-matrix interaction. The transfer of the sialic acids from cystidine-5-monophospho-N-acetylneuraminic acid (CMP-NeuAc) to the terminal position of carbohydrate group of glycoproteins and glycolipids is catalyzed by a family of sialyltransferases (STs). There is a large body of evidence to suggest that tumor cells have changed surface properties from their normal counterparts, and that these changes are partially due to altered sialo-glycoconjugates expressed on the plasma membrane and that altered sialylation (change in glycoprotein expression), which occurs during certain pathological processes, such as oncogenic transformation, tumor metastases, and invasion, is associated with enhanced ST activity. Increased β1,6-branching, increased Sialyl-Lewis epitopes, increased Sialyl-Tn antigen (Sialyl α GalNAc-O-Ser/Thr), or the general increase in sialylation of cell surface glycoproteins are commonly observed in N-lined and O-lined oligosaccharides of carcinoma cells. Carbohydrate changes occur common in breast cancer, colorectal cancer, lung cancer, hepatic carcinoma, gastric carcinoma, head and neck squamous cell carcinoma, brain tumor, choriocarcinoma, and prostate cancer. More specifically, these changes in glycoprotein expression are reported to play important roles in tumor grade, invasion, metastatic ability, and poor clinical outcome. Some of these studies also evaluated the mRNA expression of STs and found that increased mRNA expression of STs is correlated with poor outcome in breast cancers, and colon carcinoma. There are some studies that only evaluated the relationship between the change of ST mRNA expression and their clinical correlation, including colorectal cancer, and gastric cancer. In Taiwan, carcinoma of the cervix is still the most common female cancer in Taiwan with an incidence rate of 32.1 per 100,000. The prognosis is dependent mostly on many factors; among them, the majority is based on histo-pathological parameters. Many pathological factors including bulky tumor size, poor differentiation, presence of lymph-vascular space involvement, presence of parametrial invasion, and deep stromal invasion are related with lymph node metastases and closely respond to disease-free survival. Besides these conventional pathological parameters for predicting prognosis in an early stage cervical cancer, there are many biological factors (cellular molecules), which show close correlation with aggressive and invasive behaviors in tumors. Among these biological factors, sialic acids are one of the most promising molecules because they involve cell-cell and cell-matrix interactions and cellular recognition. However, so far, altered expression of mRNA of STs in gynecological cancers is still unavailable. In addition, in our laboratory (Department of Biochemistry and Institute of Biochemistry, National Yang-Ming University), Dr. Wu guided by the professor Tsai (the Director in the program of my Ph.D. in Institute of Clinical Medicine, National Yang-Ming University) in his Ph.D. program used the screening strategy to find a very specific ST3Gal I inhibitor- soyasaponin I (Ki = 2.3 μM), which is 20-fold and 25-fold higher affinity to the active site enzyme than nature substrate CMP-Neu5Ac (KM = 46 μM) and product CMP (Ki = 55 μM), respectively. Soyasaponin I contains no significant inhibition activity for other glycosyltransferases and glycosidases, respectively. This unique finding provides a new feasible method to directly elucidate the functional roles of STs by inhibition ST activity under in vivo experiments and highlight the possibility of use the biological inhibitor for cancer prevention and/or treatment. Therefore, I selected this promising topic- altered expression of sialyltransferases in cervix squamous cell carcinoma and their clinical correlation as the study project for my Ph.D. program when I entered the Institute of Clinical Medicine, National Yang-Ming University since 1999, partly because the squamous cell carcinoma of the cervix is still the most common female cancer in Taiwan, partly because the clear-cut and long-term transformation from normal tissues of the cervix, to pre-cancer lesion (cervical intraepithelial neoplasia) and further toward the invasive squamous cell carcinoma of the cervix. Of the most importance, we manage two hundred women with the early-stage squamous cell carcinoma of the cervix (FIGO IB-IIA), which is treated with radical hysterectomy and pelvic lymph node dissection in our department (Department of Obstetrics and Gynecology, National Yang-Ming University based Taipei Veterans General Hospital annually. Based on the basic support from the Institute of Biochemistry (The director; Professor Tsai) and the clinical support from the Department of Obstetrics and Gynecology (The director: Doctor Yuan & The Section Chief: Doctor Chao), I can study the change of different-type sialyltransferases in cervical normal tissues and cervical squamous cell carcinoma first. Then, I can compare the difference between the normal tissues and cancer tissues to search for candidate type of sialyltransferases, and use the same strategy to evaluate the clinical correlation between their changes and the tumor behavior such as deep stromal invasion, lymph-vascular space involvement, parametrial invasion and lymph node metastases in clinical practice. Our goal was to clarify their roles in tumor carcinogenesis of cervical squamous cell carcinoma, and their correlation in clinically poor prognosis. We attempted to use these biochemical parameters combining with histo-pathological parameters to identify the high-risk women with recurrence or therapeutic failure. After confirming their roles in cervical cancer, we can highlight the possible role in cancer prevention and treatment using these specific and selective ST inhibitors in the future. The thesis is divided into four parts: first, to set up the study method including using substrate-enzyme assay for sialyltransferase activity, and letting blotting for measuring amounts of sialic acids on the cell line; second, to use semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) for evaluating the altered mRNA of sialyltransferases in cervical squamous cell carcinoma compared to those in normal cervical tissue; third, to use the above-mentioned methods to evaluate their changes in cervical squamous cell carcinoma to clarify their relationship between conventionally clinico-pathological parameters and expression of sialyltransferases and set up the possible value for clinical practice in the future. In the program part one, we successfully detected the activities of each subtype of sialyltransferases using Galβ1,3GalNAc-acetyl-lactosamine)-Obzl (acceptor for ST3Gal I), Galβ1,3GlcNAcβ1,3Galβ1,4GlcNAc (acceptor for ST3Gal III), Galβ1,4GlcNAc (acceptor for ST3Gal IV), asialo-bovine submaxillary mucin (acceptor for ST6GalNAc I), asialo-fetuin (acceptor for ST6GalNAc II), and fetuin (acceptor for ST6GalNAc III), respectively; we also successfully detected the amounts of sialic acids using fluorescein-conjugated Sambucus nigra agglutinin (SNA) specific for α2,6-sialic acids and fluorescein-conjugated Maackia Amurensis agglutinin (MAA) specific for α2,3-sialic acids. We found that increasing enzyme activity of ST3Gal I and ST6GalNAc II might be important in various kinds of gynecological cancers. More specifically, enhanced activity of sialyltransferases involving α2,6-sialic acid sugar chains might be more important in cancer development. Future studies will investigate whether the enzyme activity of these sialyltransferases can be helpful for clinical practice. Based on these findings, we entered the program part two. We directly switched to study the difference of mRNA expression of 4-type sialyltransferases (ST3Gal I, ST3Gal III, ST3Gal IV and ST6Gal I) between the cervical squamous cell carcinoma and the normal cervical tissue using semiquantitative reverse transcription-polymerase chain reaction method and found that sialyltransferase ST6Gal I expression was enhanced in squamous cell carcinoma of the cervix (p=0.026, Mann-Whitney U test), but mRNA expression from the other three sialyltransferases (ST3Gal I, ST3Gal III, and ST3Gal IV) was significant down-expression in squamous cell carcinoma of the cervix compared to the normal cervix (p=0.003, p<0.001, p=0.001, respectively). High ST6Gal I expression was associated with more invasive property of cervical cancer, such as deep stromal invasion, lymph-vascular space involvement, and poor differentiation (p=0.010, p<0.001, p<0.001, respectively). The conclusion of the study shows that combination of enhanced sialyltransferase ST6Gal I mRNA expression and decreased mRNA expression from ST3Gal I, ST3Gal III, and ST3Gal IV might be important processes in cervical cancer. Therefore, our goal of the next study, the program part three, will investigate whether RT-PCR detection of the expression of these enzymes can be helpful for prognostic purposes. In the program part three, this study further investigates their changes in mRNA expression of the four STs in FIGO stage IB1 squamous cell carcinoma to assess the extent of sialylation associated with lymph node metastases. We evaluated the alterations in ST mRNA expression in FIGO IB1 cervical squamous cell carcinomas using RT-PCR and substrate-enzyme assay methods. We found that both ST6Gal I mRNA and ST3Gal III mRNA expressions were significantly increased in patients with lymph node metastases compared to those without lymph node metastases (p=0.002 and p=0.001, respectively, Mann-Whitney U test). Using receiver operating characteristic (ROC) curves of ST ratio index for accuracy comparison of lymph node metastases, ST3Gal III and ST6Gal I were observed to be fairly interchangeable (area under the curve (AUC) of 3Gal I=0.810; AUC of 6Gal I=0.786, significance of difference between AUC=0.810). High ST6Gal I expression was associated with other invasive properties of cervical cancer, such as deep stromal invasion and presence of lymph-vascular space involvement. ST6Gal I expression seemed to be more enhanced in bigger tumors. Our conclusion suggested that ST3Gal III and ST6Gal I were of importance for the lymph node metastases in FIGO IB1 cervical cancer patients; more specifically, over-expression of ST6Gal I was of crucial relevance for the presence of poor-prognostic factors, such as deep stromal invasion and lymph-vascular space involvement and lymph node metastases. In the program part four, based on the fact that increasing messenger ribonucleic acid (mRNA) expression of ß-galactosideα2,6-sialyltransferase I (ST6Gal I) is important in squamous cell carcinoma (SCC) of the cervix. In many tissues, ST6Gal I is transcriptionally regulated through the use of different promoters that originate in the mRNAs which diverge in the 5’-untranslated regions. In this study, to clarify the roles of ST6Gal I mRNA species in cervical SCC, we investigated their expression including a “constitutive” promoter- placental form (Y + Z form), “hepatic” promoter and a specific lymphoblastic promoter (X form) in normal and SCC tissues of the cervix using real-time quantitative reverse-transcription-polymerase chain reaction (RT-PCR). Expression of the ST6Gal I species was investigated in normal cervices (n = 38) and FIGO IB1 cervical SCC (n = 38) by real-time quantitative RT-PCR, using primers designed for amplification of a portion of the coding region common to all mRNA species, or ones for amplification of the placental transcript (Y + Z form), the hepatic transcript (H form) or lymphoblastic transcript (X form). Our results showed that ST6Gal I mRNA expression was significantly increased in cancerous tissues compared to that in normal tissues (p = 0.004, Mann-Whitney U test). Expressions of the Y+Z form did not appear to be affected by cancer transformation, since it was detected at comparable levels in normal and cancerous tissues (p = 0.13), but the H form expression was significantly enhanced in cancerous tissues compared to that in normal tissues (p < 0.0001). Surprisingly, the X form could be detected in a portion of patients either with or without cancer, but the detection rate was significantly higher in patients with cancers compared to those without cancers (86.8% vs. 52.6%, p= 0.021, Fisher’s exact test), although the X transcript was only detected at a very low level compared to the H and Y + Z transcripts. Therefore, we concluded that an increased levels of hepatic transcripts might be very important in cancer transformation because it contributes to enhanced ST6Gal I expression in cancerous tissues. A high percentage of the X form transcript in cancerous tissues might resulted from lymphocyte infiltration, and may involve a host-tumor interaction, although the clinical evidence for this is not clear yet. According to all findings in this project of Ph.D.- altered expression of sialyltransferases in cervix squamous cell carcinoma and their clinical correlation, we can clearly identify the important and significant role of altered sialyltransferase expression in the cervix squamous cell carcinoma. ST6Gal I overexpression is a very important process during the establishment of cervix SCC. With progressively growth and invasive process, enhanced ST6Gal I expression became more importance. This ST6Gal I overexpression mainly results from the activating 5’ untranslated H form promoter although both Y + Z and H transcripts contribute the total pool of ST6Gal I. When entering the metastatic status, initiated overexpression of ST3Gal III should be occurred, because overexpression of both ST6Gal I and ST3Gal III could predict pelvic lymph node metastases accurately in FIGO IB1 sqamous cell carcinoma of the cervix. We highlight the vision that, with advancing biotechnology, more specific molecules against different kinds of STs might be found in the near future, just like soyasaponin I, which is a potent and specific sialyltransferase inhibitor for ST3Gal I and is first identified in our laboratory. Using this strategy might provide a vision of possibly synergistic therapy in cervical cancer patients or offer an effective tool in cancer prevention, especially for cervix squamous cell carcinoma.

碩士
國立中央大學
化學研究所
96
Part I: Synthesis of modified lithocholic acids: coumarin, nitro-2,1,3-benzoxadiazole and other functional groups as effective anticancer agents and biological probes. Overexpression of sialyltransferase is a vital biological process, which plays a role in various physiological and pathological events, such as cell-cell adhesion, tumor cell metastasis and invasions. To increase the inhibitory potency of sialyltransferase, we prepared the coumarin, NBD (7-nitro-2,1,3-benzoxadiazole) and other functional derivatives of lithocholic acid based on our previous studies. Some of these compounds, 5-25, represent the first examples of nanomolar range sialyltransferase inhibitors with cell permeability which had been demonstrated by the experiment of wound healing and the detection of fluorescence imaging of cells incubated with coumarin or NBD derivative of lithocholic acid. Except the property of antimetastasis in wound healing assay, compounds 5-25 also slightly displayed inhibition of the proliferation of breast cancer cells with the inhibitory percentages ranging from 4~40% under the compound dose at 20 ?M. To investigate the capability of lithocholic acid derivatives attracting the sialyltransferase proteins in vitro and in vivo, nanoparticle-based sialyltransferase inhibitors, 32-36, were prepared to tackle this problem. Part II: Analogues of Malyngamide 1 and Isomalyngamide A as inhibitors of sialyltransferases: A structure activity relationship study of chain-length and functional dependence. Malyngamide 1 and Isomalyngamide A are natural products from Marine and display the significant cytotoxic activity against breast cancer cells at the low micromolar level (IC50 = 12.7 and 2.8 μM). Meanwhile, one of them also inhibited alpha-2,3-sialyltransferase activity at micromolar concentration (IC50 = 66 μM). To find out the pharmacophore, several substructures of malyngamide 1 were synthesized, and their inhibitory properties of sialyltransferase and cytotoxic activities against breast cancer cells were also studied.

碩士
國立陽明大學
生物化學研究所
88
ialic acids, a group of characteristic sugar molecules, occur at the terminal positions of the carbohydrate groups of glycoproteins and glycolipids. Some interesting relations between the amounts of sialo-glycoconjugates and embryo developments, cell differentiations, carcinogenesis, or tumor metastasis have been identified. From the viewpoint of glycobiology, cancer is the disease caused by abnormal glycosylation. The patterns of glycosylation are quite different in the transformed cell, either incomplete or over-glycosylated glycoconjugates have been observed. During the process of abnormal glycosylation of the tumor cell, there is an obvious change in the amount of surface sialic acid. In the first section of this study, for the interest of the high specific inhibitor of alpha-2,3 sialyltransferase (ST3Gal I), we developed our STI (sialyltransferase inhibitor) by doing the microorganisms extracts screening. 3,520 microbe samples were tested, we found 17 fungus, 21 bacteria and 24 actinomyces extract exist the ST inhibition activity. Further experiments are underway, including the purification of novel sialyltransferase inhibitors from several microorganisms; carcinogenesis model and tumor metastasis model in mice will be applied for analyzing the in vivo effect of these sialyltransferase inhibitors. The second section, we have focused on the investigation of the sialyltransferase activities in the gynecological cancer cell lines. In this study, the activities of each subtype of sialyltransferase and the amounts of sialic acid of different linkage type were measured in 10 gynecological cancer cell lines. The correlation between the sialyltransferase activities and the cell oncogenesis was there analyzed. Our data showed that the activities of 2 sialyltransferase subtypes, ST3Gal I & ST6GalNAc II, were significantly higher in 10 gynecological cancer cells compared to 2 normal control cells. Further experiments will be required for the understanding the roles of these 2 ST, especially ST6GalNAc II, which they play in the transformation or metastasis processes of gynecological cancers.

Podczas rozwoju choroby nowotworowej komórki budujące guz nabywają zdolności do migracji, adhezji oraz tworzenia nowych ognisk chorobowych w odległych od guza pierwotnego tkankach. Transformacja nowotworowa jest procesem złożonym, pociąga za sobą zmiany genotypu oraz fenotypu komórek transformowanych. Na powierzchni komórek nowotworowych pojawia się osobliwy zestaw białek adhezyjnych, molekuł odpowiedzialnych za utrzymanie spójności tkanki oraz prawidłową komunikację z macierzą zewnątrzkomórkową. Jednocześnie może dochodzić do nadekspresji terminalnie zlokalizowanych kwasów sjalowych wiązanych $\alpha2-3Gal$, $\alpha2-6Gal/GalNAc$ oraz $\alpha2-8SA$, co wpływa na rozwój choroby. Szczególnie interesujące z punktu tworzenia przerzutów są obserwacje, dotyczące wpływu oddziaływania integryny $\alpha5\beta1$ z fibronektyną na adhezję, migrację i inwazję komórek nowotworowych, jednocześnie obecność kwasów sjalowych na powierzchni receptora może modyfikować powyższe oddziaływania. Wyniki prezentowane w pracy dotyczą wpływu kwasów sjalowych na oddziaływania komórek czerniaka skóry pierwotnej linii WM115 oraz metastatycznej linii WM266-4 uzyskanych od tego samego pacjenta z fibronektyną. W pierwszej części rozprawy doktorskiej stosując metodę RT-PCR dokonano analizy ekspresji genów dla pięciu sjalilotransferaz przenoszących kwasy sjalowe na N-glikany. Wyniki potwierdzają, że transformacji czerniaka skóry towarzyszą zmiany w ekspresji genów kodujących sjalilotransferazy. W komórkach linii WM266-4 zwiększa się ekspresja SIAT1, zmniejsza ekspresja SIAT4c, jednocześnie ekspresja SIAT3 oraz PST nie uległa zmianie. W komórkach linii WM266-4 zaobserwowano również brak transkryptu dla STX, co może być wyznacznikiem komórek metastatycznych czerniaka skóry. W następnej kolejności badano obecność integryny $\alpha5\beta1$ stosując immunodetekcję, jak również podjęto próbę określenia sjalilacji podjednostek integrynowych metodami precypitacji lektynami oraz desjalilacji. Badania wykazują obecność integryny $\alpha5\beta1$ w komórkach obu linii czerniaka skóry jak również wzrost sjalilacji $\alpha2-6Gal/GalNAc$ oraz spadek sjalilacji $\alpha2-3Gal$ podjednostki integrynowej $\alpha5$ towarzyszy rozwojowi czerniaka. Badanie powierzchni komórek za pomocą cytometrii przepływowej wykazały zwiększoną ekspresję integryny $\alpha5\beta1$ oraz kwasów sjalowych $\alpha2-3Gal$ i $\alpha2-6Gal/GalNAc$ wiązanych do glikanów komórek linii WM266-4. Wyniki testu adhezji oczyszczonej integryny $\alpha5\beta1$ do fibronektyny wykazały, że sjalilacja $\alpha2-3Gal$ obniża adhezję badanego receptora w komórkach linii WM115 w odróżnieniu do komórek linii WM266-4, w których kwasy sjalowe nie mają wpływu na adhezję do fibronektyny. Dokonano również identyfikacji metaloproteinaz metodą zymografii. Transformacji nowotworowej towarzyszy zwiększone wydzielanie metaloproteinazy Pro-MMP-2 i pojawienie się MMP-9, co potwierdzają badania pożywki znad komórek WM266-4. W testach funkcjonalnych badano udział integryny $\alpha5\beta1$ oraz kwasów sjalowych wiązanych $\alpha2-3Gal$, $\alpha2-6Gal/GalNAc$ oraz $\alpha2-8Sia$ testem (i) gojenia ran komórek po fibronektynie, (ii) podczas adhezji komórek do fibronektyny oraz (iii) inwazji przez Matrigel komórek linii WM115 oraz WM266-4. Otrzymane wyniki wykazały, że obecność integryny $\alpha5\beta1$ zwiększa tempo migracji tylko przez komórki pierwotnej linii czerniaka WM115. Kwasy sjalowe wiązane $\alpha2-3Gal$ oraz $\alpha2-6Gal/GalNAc$ zwiększają tempo migracji komórek czerniaka skóry metastatycznej linii WM266-4. Komórki linii WM266-4 wykazują większą adhezję do fibronektyny niż komórki pierwotnej linii WM115. Integryna $\alpha5\beta1$ zwiększa adhezję komórek obu linii fibronektyny, jednocześnie ekspresja SA $\alpha2-3Gal$ oraz SA $\alpha2-6Gal/GalNAc$ jest niezbędna podczas adhezji komórek czerniaka linii WM266-4 do fibronektyny. Wyniki badania dotyczących inwazji wykazują, że komórki linii pierwotnej WM115 oraz metastatycznej WM266-4 wykazują podobną zdolność do inwazji przez Matrigel. Integryna $\alpha5\beta1$ zwiększa inwazję komórek obu badanych linii, jednocześnie podczas transformacji nowotworowej prawdopodobnie dochodzi do zmiany funkcji kwasów sjalowych. Badania wskazują, że kwasy sjalowe wiązane $\alpha2-3Gal$ sprzyjają inwazji komórek pierwotnej linii WM115, natomiast ich ekspresja nie wpływa na inwazję komórek linii WM266-4. Dodatkowo obecność kwasów sjalowych wiązanych $\alpha2-6Gal/GalNAc$ utrudnia inwazję tych komórek przez Matrigel.
During the development of cancer, cells acquire the capacity for migration, adhesion, and forming of new metastasis in distant tissues. Malignant transformation is a complex process, which involves changes in genotype and phenotype of transformed cells. On the surface of tumor cells appears a singular set of adhesion molecules, like integrin $\alpha5\beta1$, responsible for maintaining the consistency of the tissue and proper communication between cancer cells and extracellular matrix. Sialic acids are most commonly added to N-linked glycans in an a2-3, a2-6 or a2-8 linkages and altered profile of cell surface sialylation is a well-accepted hallmark of malignant transformation. The interaction of cells expressing integrin $\alpha5\beta1$ and fibronectin results cancer cell adhesion, migration, and invasion. Presence of sialic acids may modify these interactions. The aim of this study was to determine the impact of sialic acids on interactions between primary WM115 and metastatic WM266-4 melanoma cell lines and fibronectin. The aim of the present study was to analyze gene expression for five sialyltransferases witch added sialic acids to the terminal positions of N-glycans, using RT-PCR method. The results confirmed that expression of sialyltransferase genes are changing during cancer transformation. Upregulation of SIAT1 gene, downregulation of SIAT4c gene in WM266-4 metastatic cell line were observed. In addition lack of transcript for the STX was observed, which may be the determinant of metastatic melanoma cells. Subsequently, the presence of the integrin $\alpha5\beta1$ has been studied using immunodetection. The integrin subunit sialylation was investigated using enzymes digestion as well as lectin precipitation. The results revealed the presence of integrin $\alpha5\beta1$ in cancer cell lysats of both melanoma cell lines. Studies have also shown an increase $\alpha2-6Gal/GalNAc$ sialic acids and decrease of $\alpha2-3Gal$ sialic acids linked to $\alpha5$ integrin subunit. The impact of sialic acid $\alpha2-3Gal$, $\alpha2-6Gal/GalNAc$ and $\alpha2-8Sia$ linked to purified $\alpha5\beta1$ integrin receptor on protein adhesion to fibronectin was investigated using specific sialidases. The results have shown that sialic acid $\alpha2-3Gal$ have decreased integrin adhesion to fibronectin in WM115 cell line. Sialic acids linked to $\alpha5\beta1$ isolated from WM266-4 cell line had no effect on adhesion to fibronectin. Densytometric analysis of gelatin zymography showed increase expression of Pro-MMP-2 and de novo synthesis of MMP-9 by WM266-4 cell line. Expression of Pro-MMP2 and MMP-9 were associated with melanoma progression. Finally the impact of $\alpha5\beta1$ integrin and sialic acids $\alpha2-3Gal$, $\alpha2-6Gal/GalNAc$ and $\alpha2-8Sia$ linked to N-glycans was investigated during (i) wound healing on fibronectin, (ii) adhesion to fibronectin, and (iii) cell invasion by Matrigel in WM115 and WM266-4 cell lines. The results revealed that the presence of $\alpha5\beta1$ integrin increased wound healing of WM115 cell line, but sialic acids linked to N-glycans had similar effect on WM266-4 cell lines. The WM266-4 cells also showed increased adhesion to fibronectin in comparison with primary WM115 cell line. Increased cell adhesion to fibronectin is probably associated with cutaneous melanoma transformation. Integrin $\alpha5\beta1$ was also associated with increased cell adhesion in primary and metastatic cell lines. Sialic acids $\alpha2-3Gal$ and $\alpha2-6Gal/GalNAc$ expression were necessary during WM266-4 adhesion to fibronectin. Study of cell invasion indicated that primary and metastatic WM115 WM266-4 cell lines exhibited similar invasive ability threw Matrigel. Expression of $\alpha5\beta1$ integrin enhanced cell invasion in both tested cell lines. Moreover, presumably there is a change in the function of sialic acids during cell invasion. Sialic acids $\alpha2-3Gal$ promoted cell invasion of primary cell line WM115 but did not affect invasion of the in melanoma cell line WM266-4 through Matrigel. In addition sialic acids $\alpha2-6Gal/GalNAc$ reduced invasion of WM266-4 cells through Matrigel.

博士
國立陽明大學
生物化學研究所
88
A number of reports illustrated that sialyltransferase is significantly altered during normal development and differentiation, oncogenic transformation, and the metastasis potential. Therefore, it is highly necessary to discover the potent sialyltransferase inhibitors to elucidate the influence of alteration of sialyltransferase expression in the complex biological systems. Up to now, no effective cell membrane-permeable sialyltransferase inhibitor has been developed to use in cell culture system. Therefore, in the thesis, I focus on the discovery of the novel sialyltransferase inhibitors, which can be applied to sialobiology. First, the sialyltransferases were constructed and expressed in mammalian cells. WCY have been discovered to be a novel sialyltransferase inhibitors. It highly specific inhibition for sialyltransferase but not for other glycosyltransferases and glycosidases. For in vivo activity, WCY present the inhibition activity for reducing the expression level of cell surface sialoglycoconjugates. These results indicate that WCY is the first sialyltransferase inhibitor which can be used to inhibit the expression of sialoglycoconjugates in the cell level, and it also provides a new information to design the more potent inhibitor of sialyltransferase to elucidate the functional roles of sialyltransferase and sialoglycoconjugates. 英文摘要 壹、簡介- - - - - - - - - - - - - - - - p.1 貳、實驗材料 - - - - - - - - - - - - - - p.13 參、實驗方法- - - - - - - - - - - - - - p.15 肆、結果- - - - - - - - - - - - - - - - p.30 伍、討論- - - - - - - - - - - - - - - - p.48 陸、總結 - - - - - - - - - - - - - - p.61 柒、參考資料- - - - - - - - - - - - - - p.62 捌、表- - - - - - - - - - - - - - - - p.70 玖、結果圖- - - - - - - - - - - - - - - p.81 拾、論文(1)- - - - - - - - - - - - - - - - p.118 拾壹、論文(2)- - - - - - - - - - - - - - - p.136 拾貳、附圖- - - - - - - - - - - - - - - p.148

博士
國立陽明大學
生化暨分子生物研究所
94
Sialic acids belong to a nine-carbon amino sugar neuraminic acid family, and they are widely distributed in nature as the terminal sugars of oligosaccharide chains of glycoconjugates (glycoproteins and glycolipids). Recently, it has been demonstrated that the sialylation on cell surface is involved in biological and pathological processes, such as differentiation, oncogenic transformation and tumor metastases and invasions. Cell sialylation is dependent on the sialyltransferase which catalyst the transfer of sialic acid from cytidine-5-monophospho-N-acetylneuramic acid (CMP-Neu5Ac) to the terminal position of oligosaccharide chain of glycoconjugate. Therefore, modulation of the sialyltransferase (ST) activity has become an interesting and important subject in the elucidation of the roles played by ST either in normal or pathological processes. In 1980s, although the ST inhibitors based on the analogs of donor substrate were successfully synthesized, these compounds showed no substrate specificity and could not be used in clinical application. Recently, lead compounds based on the transition-state structure of CMP-Neu5Ac have been tested successfully and achieved good inhibition in vitro. However, these transition-state structure-based inhibitors are cell-impermeable and not suitable for in vivo applications. Therefore, in this thesis, we tried to find out new ST inhibitors from natural products and microbial metabolites for potential in vivo application. Sixty-two candidates were screened out from 3520 samples and 9 strains of fungi, 10 strains of bacteria, and 9 strains of actinomyces were obtained. We also tentatively tried to purify the ST inhibitor from the fungus strain 132359 and about 3 g of crude product was successfully obtained from the culture media by using the submerged fermentation method. In 2001, we discovered a new ST inhibitor, soyasaponin I (SsaI), from the soybean extract. In this thesis, the effects of SsaI on the metastatic and invasive behaviors of several cancer cells were also studied. At first, the cytotoxic effect of SsaI on cancer cells were investigated and the results showed that SsaI displayed significant growth inhibition activity only at concentration greater than 100 μM. Then, the ST enzyme activities of various cancer cell lines were measured and it showed that MCF-7 cell has high ST3Gal I enzyme activity; therefore, the MCF-7 cell were chosen for further analysis. The α2,3-ST activity in cellular homogenate prepared from MCF-7 cell could be inhibited by SsaI. When added in culture medium, the SsaI not only reduced the amount of α2,3-sialic acid on MCF-7 cell surface in a dose-dependent manner but also stimulate the adhesion of MCF-7 cell to both type I collagen and the Matrigel-matrix. SsaI also significantly decreased the migration ability of the highly metastastic MDA-MB-231 cell. All together, these studies suggested that SsaI could potentially be used to affect the invasive behavior of tumor cells by modulating the activity of ST. These data suggest that de-regulated α2,3-sialylation could played a crucial role in the invasion and metastases of tumor cells. SsaI is a good candidate for studying the biological roles of ST, and might provide a new preventive strategy in tumor metastasis.

碩士
國立中央大學
化學研究所
98
Nanoparticles bearing surface-conjugated specific inhibitors are increasingly being utilized for a number of bio-applications including identification, separation and enhancement of desired proteins. These attractive bio-applications involve specific adsorption and recognition; however, these are the major hindrances for nanobiotechnology. To solve this question, we have investigated the effect of nanoparticle surface displaying high affinity molecules, such as sialyltransferase inhibitors. Maintaining a reasonable affinity toward desired protein is generally a prerequisite for proper design of nanoparticle-conjugated specific inhibitors. We prepared sialyltransferase inhibitors, lithocholic acid derivatives with L-Asp or NBD-L-Asp moiety, which have IC50 values at micromolar ranges. Next, magnetic nanoparticle (iron oxide)-conjugated lithocholic acid derivatives, compounds 15~16, were synthesized via click chemistry. Herein, we demonstrated that compounds 15~16 have the ability to identify, separate and enhance binding to the target alpha-2,3(N)-sialyltransferase, a glycol- and membrane-bound protein. The protein band (38 kDa) on SDS-PAGE derived from rat was confirmed by LC MS-MS analysis and proteomics searching. Extending these studies to crude cell lysate in vitro experiment, we found several interesting proteins including talin. Further investigation of talin toward metastasis in cancer is in progress.

博士
國立陽明大學
生化暨分子生物研究所
94
The transfer of sialic acids to the non-reducing terminal positions on sugar chains of glycoconjugates is catalyzed by sialyltransferases (STs). These sialylated glycoconjugates not only have a role in providing structural components, but also in mediating cell-cell and cell-microbe interactions. Additionally, increased sialylation is correlated with oncogenic transformation and metastatic potential. Therefore, ST inhibitors may be potentially valuable as anticancer and antimetastatic agents. Previously, our laboratory has found a ST inhibitor, soyasaponin I (Ssa I) from soybean extract. Ssa I can reduce the expression of the alpha2,3-linked sialic acids on B16F10 melanoma cells, and inhibit the migration ability of B16F10 cells. This study demonstrated that Ssa I only specifically inhibited the expression of alpha2,3-linked sialic acids without affecting other glycans on B16F10 cells. Moreover, Ssa I can enhance cell adhesion to extracellular matrix proteins. A pulmonary metastasis assay demonstrated that alteration of glycosylation in this way significantly reduced the ability of tumor cells to distribute to the lungs of mice. Collectively, these findings suggested that alpha2,3-linked sialic acids may play an important role in metastasis potential of B16F10 cells. Additionally, through screening of microbial extracts for ST inhibitors in our laboratory, we found that the extract of fermentation broth from Pseudomonas aeruginosa 5565 had strong inhibitory activity. The active compound was purified and identified as the mixture of two rhamnolipid homologs, 2-O-alpha-L-rhamnopyranosyl-alpha-L-rhamnopyranosyl-beta-hydroxyldecanoyl-beta- hydroxyldodecanoate (RhaRhaC10C12) and 2-O-alpha-L-rhamnopyranosyl-alpha-L-rhamnopyranosyl-beta-hydroxyldodecanoyl-beta-hydroxyldecanoate (RhaRhaC12C10). RhaRha(C10C12) only specifically inhibited ST activity, without affecting fucosyltransferases and galactosyltransferase activity. Kinetic analysis indicated that RhaRha(C10C12) is a competitive inhibitor with respect to the donor CMP-Neu5Ac and a noncompetitive one with respect to the acceptor substrate LacNAc with Ki of 25.7 and 16.3 mM, respectively. The commercial rhamnolipid mixture (JBR599) was used to treat A549 lung cancer cell to determine whether JBR599 altered A549 cell surface glycosylation, the result showed that the expression of the a2,3-linked sialic acids on treated cell surface was reduced significantly. On the contrary, the expression of the asialoGM1 was increased significantly. P. aeruginosa is a major pathogen in lung bacterial colonization. It can adhere to epithelial cells via pili, which recognize a GalNAcbeta1-4Gal disaccharide, exposed in asialylated glycoconjugates such as asialo GM1. Our results also demonstrated that the number of adherent P. aeruginosa on rhamnolipid-treated A549 cells was increased compared with control. It suggests that chronic exposure to rhamnolipids may alter the glycans profile on host cells, and subsequently enhance P. aeruginosa adhesion to host cells and spreading in vivo.

碩士
國立中正大學
化學暨生物化學研究所
101
According to many reports, abnormal expression of the sialyltransferase has been found to actually connect with many cancers. Therefore,purposes and objectives of this study are making effort to develop effective sialyltransferase inhibitors and regulate the level of sialyltransferase expression. In the thesis, we initially synthesized potential sialyltransferase inhibitors of the fluorine-substituted lithocholic acid derivatives. Then we use lithocholic, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid hyodeoxycholic acid and cholic acid as their parent skeleton, and take a place of a hydrogen atom or hydroxyl groups to single fluorine or two fluorines. Employing the strategy of permutation and combination, twelve fluorine-substituted lithocholic acid derivatives at the C3, C7 and C12 position in the A, B, C ring of the skeleton were synthesized respectively. We next took all the flourine-substituted analogs to do the bioactivity test of wound healing and α2 ,3-sialyltransferase inhibition test. Interestingly, we found that compounds YL-38, YL-44 possess potent anti-metastasis ability and compounds YL-34, YL-72, and YL-80 could be identified as protential α2 ,3-sialyltransferase inhibitors in comparison with the parent compound, lithocholic acid.

Thesis (M.S.)--State University of New York at Buffalo, 2008.
Title from PDF title page (viewed on Feb. 11, 2009) Available through UMI ProQuest Digital Dissertations. Thesis adviser: Rittenhouse-Olson, Kate Includes bibliographical references.

碩士
國立中正大學
化學暨生物化學研究所
107
According to cause of death statistics in ministry of health and welfare, cancer is the most lethal disease. Cancer metastasis is the major cause of death in cancer patients. In previous study, overexpression of sialic acids on the tumor cell surface is a hallmark of cancer, and the upregulation of sialyltransferase activity is associated with cancer metastasis. Sialic acids are monosaccharides, found as terminal unit on the cell surface and should be recognized by siglecs on the immune cells. Sialyltransferases are one of glycosyltransferases, and can transfer sialic acids to glycoproteins or glycolipids. Building on these the results, we want to develop the sialyltransferase inhibitor to retard sialic acid transferring to the cell surface leading to. Reduction of cancer cell growth and suppression of cancer metastasis. Here, we synthesized a new and novel skeleton based on homolithocholic acid, lithocholic acid-analogue, where the structure possessed NBD group and different linkers of N3. The IC50 values of homolithocholic acid analogues are around the range of 6-14 μM against N-glycan ST6GAL1. Furthermore, they did not block the sialylation of O-glycan ST3GAL1, suggesting that this series of sialyltransferase inhibitors demonstrate their specific selectivity toward N-Glycan sialylation versus O-Glycan sialylation. In addition, homolithocholic acid analogues had cytotoxicity, when MTT assays tested under the culture condition without serum. The results of anti-migration effects derived from homolithocholic acid analogues indicated that cytotoxicity is the main reason for the action of mechanism.

碩士
國立中央大學
化學研究所
91
Part 1 A new system for hydrogenation of haloalkenes is reported. Cu(II)/Fe(III)-mediated selective hydrogenation of steroidal haloalkenes in the presence of hydrazine proves to be a very efficient method for the synthesis of 17b-halosteroids, potential candidates as antiestrogens or androgen receptor-mediated imaging agents. The reaction stereo- specifically affords b-haloalkanes without any concomitant formation of dehalogenation products. Part 2 A new approach to the synthesis of anomeric sulfur analogues of CMP-Neu5Ac containing alkane or arene linkage is described. The procedure involves the high b-stereoselectivity in sialylation of the peracetylated sialic acid methyl ester with mercaptoalkyl (aryl) trichloroacetate, followed by selective deprotection of the trichloroacetyl group to the corresponding hydroxyalkyl and hydroxyaryl thioglycosides. Subsequent O-phosphitylation, oxidation and deprotection led to the isolation of the target compounds.

碩士
國防醫學院
牙醫科學研究所
106
Background: The expression of Tn/STn in inflamed tissue had been discussed before. In our previous experiment, we have found the different expression ratio of Tn/STn in experimental periodontitis rat model. The expression of related transferring enzyme including sialyl-transferase, COSMC and T-synthase are investigated in this study with experimental periodontal rat model also in clinical inflamed human gingiva. Also,the possible signaling pathway is investing through LPS treated HGF(human gingival fibroblast) with inhibitors (NF-kB,ERK,JNK,PI3K,PKC). Method: Experimental periodontitis in rats was induced by either silk-ligature around the molars. The expression of sialyl-transferase, COSMC and T-synthase were examined by immunohistochemistry. In human gingiva fibroblasts (hGFs) induced by Porphyromonas gingivalis lipopolysaccharide (P.g. LPS) was treated with cell signaling inhibitors (NF-kB, ERK, JNK,PI3K,PKC).The in vitro expression of sialyl-transferase, COSMC and T-synthase was observed by real-time PCR. Results: In the ligation-induced periodontits rat model ,the expression of sialyltransferase(ST6GalNAc-1) was gradually enhanced during the period of 6 days. However,the expression of COSMC was gradually reduced through time. And T-synthase(C1GalT1) did not show an obvious correlation with ligation time. A strong positive correlation between the immunoreactivity scores of sialyl-transferase and ligature duration, but a negative correlation in COSMC was observed in the gingiva with ligature. In human gingiva samples, we also performed the IHC stainging.The sample are categorized by the potions of inflammatory cells infiltration, and were graded into 3 degrees.The expression of sialyltransferase was found with higher immunoreactivity score in severe inflamed gingiva tissue.Whereas,COSMC showed an opposite expression pattern,as the inflammatory score gain ,the expression of COSMC decreased.The expression of T-synthase did not show obvious change as the inflammatory score change. In vitro, the expression of sialyltransferase in hGFs was up-regulated after LPS treatment, and showed a highest pick after 7 hours. But the expression of COSMC gene was down-regulated,and had the lowest expression after 3 hours.The expression of T-synthase gene did not show obvious change after LPS treatment. Conclusion: The expression of COSMC decreased as the higher of the inflammatory score, which inhibits the activation of T-Synthase, and block the transformation from Tn to T antigen. Whereas, the expression of sialyltransferase which transfer Tn into STn increased as the higher grading of inflammatory score,and explained the higher expression of STn showed in our previous experiment.

碩士
國立中央大學
化學學系
103
PartI. According to many researches, abnormal expression of the sialyltransferase has been found to actually connect with many cancers. Therefore, purposes and objectives of this study are making effort to develop effective sialyltransferase inhibitors and regulate the level of sialyltransferase expression.In the thesis, the main structure of our compounds are lithocholic acid. We used high efficient and selective reagent─Selectfluor to add a fluorine atom at the C4 position as modification, and we synthesized fluorinated compound of Lith-O-Asp-NBD as effective anti-cancer compound further.Through MTT assay and Wound healing assay experiment to discuss fluorinated compound’s influence on biologically activity. Interestingly, We found that compounds JO-5b, JO-13a, JO-13b possess potent anti-metastasis ability. PartII. Fluorescent labeling has the advantage of high sensitivity and good selectivity for the use in a wide range of applications. In recent years, novel molecular imaging agents were developed for the use of clinical translation through early lesion detection, molecular targeted therapy, therapeutic monitoring, and the development of new anti-cancer drugs. This action significantly improves diagnosis and therapy in patients with breast cancer metastasis. In this thesis, the main structure of our compounds is benzoxazinone. We modified its functional group to explore the relationship between structure and fluorescent quantum yield of molecular probes. Through analysis of the optical property, we found that Jo-35 has the highest quantum yield (Φ = 0.39) compared to that of others. Intramolecular hydrogen bond was confirmed in the skeleton between oxygen atom and amino group of the benzene ring. Molecular probes drop the fluorescent property if the intramolecular hydrogen bond is absent due to replacement of the amino group by other atom and/or moiety.

碩士
國立臺灣師範大學
化學系
100
Aberrant expressions of sialyltransferases (STs) have been reported to positively correlate with many cancers. Therefore, we interest that the development of sialyltransferase inhibitors to modulate sialyltransferase activity and thus alleviate or inhibit physiological processes (e.g., alteration of sialylation in cell surface and sialylation of glycoproteins/glycolipids) caused by sialyltransferases. In this thesis, we initially designed a potential STs’ inhibitor containing an olefin moiety within C-ring (C-11/C-12) of the lithocholic acid as a parent skeleton (compound EY-22). Synthesis of EY-22 was carried out from deoxycholic acid as the starting material. First, both the carboxylic acid at C-24 and the hydroxyl group at C-3 of deoxycholic acid were selectively protected. The protected deoxycholic acid was esterified to yield the required mesylate ester (mesylation of the hydroxyl group at C-12), which was subsequently converted to the protected lithocholic acid with an olefin at C-11 by the method of demesylation. However, demesylation resulted in a mixture compounds with the presence of desired product and steroidal rearrangement compound in the ratio of 1:1. Efficient formation of demesylation product was obtained from modification of reaction conditions. The final product EY-22 was accomplished after removal of the remaining protecting groups. A series of EY-22 based derivative, EY-36、EY-37、EY-39、EY-43、EY-45 and EY-46 (containing Asp, Glu, NBD and 4-nitrobenzoic acid moieties), were successful prepared according to the procedure described previously. Interestingly, effective disruption of wound closure was observed for cells cultured in the presence of derivatives EY-36、EY-39 and EY-45 compared to deoxycholic acid and DX-5. Assays of sialyltransferase inhibition are underway.

碩士
國立中央大學
化學學系
101
Recently, aberrant sialylation is found in cancer metastasis frequently. Empolying the strategy of the relationships between structure and activity , we use sialyltrasdferase as the molecular target to develop its inhibitors in order to attenuate cancer metastasis. Preparation of Lith-O-Asp-coumarin derivatives was successfully accomplished through the coupling reaction between Lith-O-Asp and coumarin moieties using DMMTM BF4 (4-(4,6-dimethoxy-(1,3,5)triazin-2-yl)-4-methyl-morpholinium tetrafluoroborate) or SOCl2 as the reagents. Extensive SAR studies were performed using different substituted-coumarin analogs under the wound healing and sialyltransferase inhibitor assays. Among the Lith-O-Asp-coumarin derivatives, the hydroxyl-substituent analogs at C-7 position were observed to have the hightest biological activity. It is also noteworthy that the methyl-substituent analogs at C-8 position also display significantly increased biological activity toward both assays.

碩士
國立中山大學
生物醫學研究所
97
Sialyltransferases (STs), which catalyze the sialylation reaction by adding sialic acids to the terminal positions of oligosaccharide of glycoproteins and glycolipids, are over-expressed in cancer cells and associated with cancer metastasis. Until now, ST inhibitors are not applicable for clinical use because of poor cell permeability, although showing potent effect in vitro. In this study, we synthesize a lithocholic acid-based ST inhibitor AL10 and test its anti-metastatic effect. Overexpression of α-2,3-ST is found in highly metastatic A549 and CL1-5 lung cancer cells. Confocal microscopy demonstrates that AL10 is cell permeable and may attenuate total sialylation on cell surface. AL10 has no cytotoxicity but inhibits adhesion, migration, actin polymerization and invasion of A549 and CL1-5 cells in vitro. Inhibition of adhesion and migration by AL10 is associated with reduced sialylation of beta1 integrin. In addition, activation of the beta1 integrin downstream signaling molecule focal adhesion kinase is also attenuated. More importantly, AL10 suppresses lung metastasis in vivo and this effect may be linked with reduced sialylation of the chemokine receptor CXCR4 which has been found to play a critical role in organ-specific metastasis. Serum biochemical assay indicates that AL10 does not affect liver and kidney functions of experimental animals. Taken together, we conclude that AL10 is an effective sialyltransferase inhibitor and exerts anti-metastatic effect in vivo via suppression of sialylation of beta1 integrin and CXCR4.