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1
Ho, Sze-hang, and 何思恆. "Differential expression of Wnt inhibitors Dickkopf-1 (Dkk-1) and Wnt inhibitory factor-1 (Wif1) in the regulation of urorectal development." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2014. http://hdl.handle.net/10722/207999.
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In mammals, the external genitalia, urinary tract and anorectal tract are developed from a common embryonic primordium, the urorectum. Cloaca is the hollow space inside the urorectum that connects the hindgut and the urogenital sinus. During the urorectal development, the external genitalia is formed from the outgrowth of genital tubercle (GT) protruding from the urorectum, while the future urinary tract and anorectal tract are formed by the partition of cloaca during cloacal septation. GT outgrowth and cloacal septation are important developmental events for the formation of genitourinary and anorectal system. In human, dysregulation of these developmental events results in congenital anorectal malformations (ARM).
Wnt signaling is one of the key signaling pathways that regulates urorectal development. The activity of Wnt signaling is initiated by the binding of Wnt ligands to cell surface receptors, which can be antagonized by secretory Wnt inhibitors. Dickkopf1 (Dkk1) and Wnt inhibitory factor 1 (Wif1) are secretory Wnt inhibitors implicated in urorectal development. However, the functions of other secretory Wnt inhibitors during urorectal developments remain to be elucidated.
In this study, expression analyses showed that Dkk1, Dickkopf2 (Dkk2), Dickkopf4 (Dkk4), Secreted Frizzled-related Protein 1 (Sfrp1) and Wif1 were expressed in the developing urorectum. The dynamic, overlapping and restricted expression patterns of these Wnt inhibitors were closely associated with the GT outgrowth and the cloacal septation events, implying that these Wnt inhibitors functioned in a coordinated manner in defining the field of Wnt signaling activities in the developing urorectum.
Wif1 knockout mice (〖Wif1〗^(-/-)) was used as the model to investigate the functions of and the interplay between secretory Wnt inhibitors in urorectal development. GT outgrowth and cloacal septation defects were observed in 〖Wif1〗^(-/-) embryos. Most of the 〖Wif1〗^(-/-) embryos displayed varying degrees of GT outgrowth defects, while septation defects were only occasionally observed. This suggested that GT outgrowth and cloacal septation were regulated by Wif1 via different regulatory mechanisms.
In the urorectum of 〖Wif1〗^(-/-) embryos, Dkk1 was significantly upregulated in the peri-cloacal mesenchyme. Further expression analysis suggested that Dkk1 was sufficient to rescue cloacal septation defects but not GT outgrowth defects in 〖Wif1〗^(-/-)embryos. In the 〖Wif1〗^(-/-) embryos with severe GT outgrowth defects, the Fgf8-expressing distal urethral epithelium, the signaling center in the urorectum, was absent, suggesting that the GT outgrowth defects could be contributed by the loss of dUE-expressing signals such as Fgf8.
This study demonstrated the importance of secretory Wnt inhibitors in the GT outgrowth and cloacal septation and suggested that secretory Wnt inhibitors played partially overlapping roles in urorectal development. A rescue mechanism for cloacal septation performed by Dkk1 upon Wif1 deletion was proposed. Such auto-regulatory mechanism within the Wnt signaling pathway indicated that Wnt inhibitors play essential regulatory roles in the urorectal development and a balanced Wnt signaling activity modulated by Wnt inhibitors is crucial to the development of urorectum.published_or_final_version
Surgery
Master
Master of Philosophy
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Jandu, Arvinder Singh. "Characterization of Novel Canonical WNT Signaling Inhibitors in Prostate and Colorectal Cancers." Thesis, The University of Arizona, 2013. http://hdl.handle.net/10150/297656.
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Objectives: The objectives of this study were to (1) characterize the levels of WNT signaling in DLD-1 colorectal cancer cells with mutant APC and DU145 prostate cancer cells with wild type APC, (2) to conduct an investigation of novel tankyrase inhibitors in both colorectal and pancreatic cancer cells. WNT signaling has been shown to play an integral role in the proliferation and metastasis of both cancer types; therefore, it is of interest to evaluate inhibitors of the signaling pathway to assess their potential applications in targeted cancer therapeutics. Methods: To characterize WNT signaling activity in colorectal cancer cells (1), DLD-1 cells transduced with a TCF/LEF luciferase reporter and selected with puromycin were grown out in both a two dimensional culture and as tumorspheres to comparatively examine signaling between the different morphologies, as well as at different plating densities. To probe the mechanism of the tankyrase inhibitors (2), DLD-1 and DU145 pancreatic cancer cells were grown to confluency, treated with 1 uM of each inhibitor, and harvested for lysate preparation after 24 hours. Western blots of β-catenin, AXIN1, and AXIN2 were performed and quantified for comparative analysis. Additional assays were performed using DLD-1 cells to assess effects of the inhibitors on proliferation and migration. Results: In DLD-1 cells, it was observed that all tumorsphere cultures exhibited significantly decreased levels of WNT signaling. In DU145 cells, inhibitor D was demonstrated (western blot) to knock down β- catenin levels, as well as stabilize both AXIN1 and AXIN2 levels. Inhibitor A3 was able to decrease β- catenin levels, as well as stabilize AXIN2 levels. Surprisingly, inhibitor A1 was found (via densitometry) to increase both beta-catenin levels and AXIN2 levels. A2 and IWR were found to only decrease β- catenin protein amounts. In DLD-1 cells, all inhibitors exhibited some decrease in beta-catenin levels; however, only A1, A3, A5, and A6 were found to significantly decrease proliferation (MTT assay), and only A1, A4, and A7 were found to significantly stunt migration (scratch assay). Conclusions: In DU145 cells, tankyrase inhibitors D and A3 display the most promise by being able to modulate both catenin and AXIN protein levels. In DLD-1 cells, growth and proliferation assays demonstrate that some inhibitors result in differential phenotypic effects, suggesting that different gene targets downstream of β-catenin are being effected. Further blots for such targets will prove useful to clarify these results. In addition, similar phenotypic characterizations in DU145 cells are necessary to connect the tankyrase mechanisms to observable effects.
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García, Reyes Balbina [Verfasser]. "Validation of new Casein Kinase 1 (CK1) small molecule inhibitor compounds and characterization of Inhibitors of Wnt Production (IWPs) as inhibitors of CK1δ / Balbina García Reyes." Ulm : Universität Ulm, 2018. http://d-nb.info/1151938424/34.
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Ali, Youmna Tantawy Abdou Ahmed. "Molecular regulation on the activity and expression of human organic anion transporting polypeptides (OATPs) and organic anion transporters (OATs)." Thesis, The University of Sydney, 2022. https://hdl.handle.net/2123/28906.
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Organic anion transporters polypeptides and organic anion transporter are 2 main subfamilies of SLC transporters that are located all over the body. They are influx membrane transporters that govern the movement of endogenous and exogenous compounds across the cell membrane.
Endogenous compounds such as thyroid hormones, prostaglandins, and bile acids as well as exogenous compounds like antibiotics, anti-hypertensive, statins, and anti-cancer drugs can be transported by SLCs. Altered expression of OATPs and OATs leads to several diseases and impacts drug’s pharmacokinetic and pharmacodynamic properties, which may lead to variable drug responses.
In Chapter 1 we studied SLC transporters, mainly OATs’ structure, localization, function, and molecular regulation. Genetic variation of SLCs and/or molecular regulation “by manipulating different signaling pathways” significantly altered the function and expression of transporters leading to inadequate drug efficacy, drug toxicity, and /or drug-drug interaction.
Nowadays, due to the recent approach towards herbal medicine for the treatment of several diseases we studied the involvement of human organic anion transporting polypeptides (OATPs) in drug-herb/food interactions (Chapter 2).
We found that several herbal medicines and natural compounds that exist in food can impact the function of several SLC transporters. As the transporters are responsible for the uptake of several drugs this could cause drug-herb/food interactions. Increasing the awareness of these interactions could help us to improve clinical outcomes and avoid unexpected toxicities of drug therapies in patients.
Next, we studied the molecular mechanism by which drugs (Wnt inhibitors in Chapter 3), or natural compounds (PL in Chapter 4) affect the function and activity of transporters. The function and expression of OATs and OATPs are regulated by multiple signaling kinases, including protein kinase C and protein kinase A. The canonical Wnt/β-catenin signaling pathway regulates cell proliferation, differentiation, and morphology. In Chapter 3 we found that Wnt inhibitors modulated the expression and function of important OAT and OATP transporters. PRI-724 (newly developed Wnt inhibitor in clinical trials) decreased the substrate uptake by OAT4 and OATP2B1 with less pronounced decreases in OAT1, OAT3, and OAT1A2. Two other models of Wnt inhibitors - FH535 and 21H7 - were also tested in comparative studies, where they also strongly decreased the activities and membrane expression of multiple OATs/OATPs. Thus, implicating the Wnt signaling pathway in the regulation of the function and activity of OATPs and OATs.
Piperlongumine (PL) is a natural compound traditionally used for the treatment of respiratory tract infectious diseases. Recently, PL is used in novel therapies for the treatment of different kinds of cancers. It was found that PL regulates several signaling pathways (e.g., AMP-activated protein kinase) that influence the function and activity of OATPs and OATs. Therefore, in Chapter 4 we studied the regulatory effect of PL on human organic anion transporting polypeptides (OATPs) and organic anion transporters (OATs). PL selectively increased the function of OATP2B1 and OAT4. The regulatory effect of PL is mediated through upregulating the cell surface expression of OATP2B1 and OAT4 and increasing the Vmax of both transporter proteins. Altered SLC transporter’s function may impact the pharmacokinetics and pharmacodynamics of drugs, drug treatment outcomes, unexpected toxicities, and/or drug-drug interactions.
Overall, SLC transporters play a significant role in human health and disease. Studying SLCs and their molecular regulation will help us to improve the safety and efficacy of drugs as well as understanding several human diseases in which SLC transporters are involved.
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Quevedo, Camilo E. "Design and synthesis of Quinazolinone-based libraries for inhibitation of Kinase activity and hit-to-lead optimisation of Wnt pathway inhibitors." Thesis, Institute of Cancer Research (University Of London), 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.510367.
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Kragelund, Dominik [Verfasser]. "Untersuchungen der Serum-Konzentrationen des proinflammatorischen Glykoproteins wnt-5a und seines antiinflammatorischen Inhibitors sFRP-5 bei an Sepsis erkrankten Patienten im Verlauf / Dominik Kragelund." Kiel : Universitätsbibliothek Kiel, 2016. http://d-nb.info/111754060X/34.
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Park, Kyoungmin. "The characterization of PEDF's broad activity in the ocular disease." Oklahoma City : [s.n.], 2010.
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Ng, Chun-laam, and 吳圳嵐. "Wnt inhibitory factor 1 (Wif-1) coordinates Shh and Wnt signaling activities in urorectal development." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hub.hku.hk/bib/B48329629.
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In vertebrates, the urogenital sinus and the hindgut are connected at a hollow region called cloaca. A midline mesenchymal structure known as urorectal septum (urs) descends from the ventral body wall to separate the urogenital sinus from the hindgut before the formation of an anal opening. Subsequent cloaca membrane regression at the ventral midline of the genital tubercle (GT) is crucial for the formation of an anal opening. These two events are important during cloaca septation in urorectal development.
Mice with defective Shh or Wnt signaling displayed similar urorectal defects such as GT agenesis, un-partitioned cloaca (persistent cloaca) and proximal urethral opening that are attributable to increased cell apoptosis. Furthermore, Shh and Wnt signal transduction coordinate with each other and regulate cell survival of the developing urorectum. However, the molecular mechanisms by which these two signaling pathways coordinate in urorectal development remain unclear.
We previously identified Wnt inhibitory factor1 (Wif1) from Affymetrix array analysis for genes/pathways that is implicated in urorectal development. Wif1 is a secreted protein that binds directly to Wnt ligands preventing Wnts from binding to receptors. This leads to -catenin degradation and thereby inhibits their activities. It is known that Wif1 binds to Wnt3a and Wnt5a with high affinity and deletion of Wnt3a, Wnt5a and -catenin in mice caused GT agenesis, persistent cloaca and proximal hypospadias. Using ETU-induced anorectal malformations model, I found out that Wif1 is ectopically expressed in the un-tubularized and un-septated urorectum. Wif1 is mainly expressed at the fusing endoderm that associates with programmed cell death during cloaca septation. Exogenous addition of Wif1 protein in urorectum culture also caused cloaca membrane disintegration, and proximal urethral opening that may be due to aberrant apoptosis.
Shh and Wif1 are differentially expressed at the cloaca endoderm. In normal mice, Shh is highly expressed at the cloaca endoderm except those Wif1-expressing endodermal cells. Blockage of Shh pathway by cyclopamine in urorectum culture induced ectopic expression of Wif1, concomitant with genital tubercle hypoplasia and un-septated cloaca. More importantly, deletion of Shh in mice hastened Wif1 expression at the cloaca membrane endoderm and elicited increased cell death in the
Wif1 expressing endoderm. Wif1-/- embryos display urorectal defects including delayed genital outgrowth and proximal hypospadias. Therefore, disruption of spatiotemporal expression of Wif1 could lead to defective Wnt signaling and contributes to abnormal urorectal development in Shh-/- mutant.
Current study revealed that Wif1 is involved in urorectal development and is implicated in urorectal defects. It may function as a pro-apoptotic factor to regulate endodermal cell death which is essential for the septation process. Its specific expression is restricted at the midline cloaca endoderm by Shh signaling to inhibit local Wnt--catenin activities during cloaca septation. I proposed novel hypothetical models to explain (1) the significance of the tempo-spatial expression of Wif1; (2) the significance of cell death; and (3) the molecular mechanism that Shh signaling regulates Wnt signaling activities through Wif1 in urorectal development.published_or_final_version
Surgery
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Doctor of Philosophy
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Sena, Elena. "The Transcription Factor Barhl2 Inhibits Wnt Canonical Signaling during Xenopus Embryogenesis." Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS090/document.
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Le développement embryonnaire est un processus hautement contrôlé où différentes voies de signalisation se coordonnent pour la construction d'un organisme. L'une des principales voies de signalisation impliquées dans ce processus est la voie canonique Wnt. La longue quête pour comprendre la cascade de signalisation Wnt/β-catenine a révélé que la réponse transcriptionelle induite par le signal Wnt/β-catenine est dépendante du contexte, ou compétence, cellulaire. Peu de choses sont connues sur les évènements moléculaires qui influencent cette compétence cellulaire. Dans les embryons de X. laevis Wnt/β-catenine est le signal inducteur pour l'Organisateur de Spemann. On ne sait pas ce qui limite l'activité Wnt dans ce territoire et par voie de conséquence la taille de l'Organisateur. Les résultats présentés dans ce manuscrit de thèse montrent que le facteur de transcription Barhl2 affecte le développement de l'organisateur de Spemann. Nous démontrons que Barhl2 inhibe l'activité Wnt via son interaction avec le corépresseur Groucho et le facteur de transcription Tcf, et mobilise l'activité de Hdac1 qui agit sur la structure chromatinienne. En utilisant des expériences in vitro et in vivo sur des cellules en culture et des embryons de Xénope nous démontrons que la régulation de Barhl2 sur les activités Groucho-Tcf est maintenue pendant l'embryogenèse et joue un rôle dans le confinement des progéniteurs neuraux dans le cerveau. Ensemble, nos résultats fournissent un mécanisme nouveau et important agissant sur le contrôle de l'activité transcriptionelle Wnt et la compétence des cellules à répondre à ce signalEmbryonic development is a highly controlled process where different signaling pathways participate into the elaboration of an organism. One of the main signaling pathways is the Wnt canonical pathway. The long-lasting search to understand Wnt/β-catenin transduction cascade revealed that the net transcriptional read out of Wnt/β-catenin signaling is highly dependent on the cellular context. In X. laevis embryos Wnt/β-catenin signaling is the informative signal for the Spemann Organizer induction. However little is known on what limits Wnt activity in this territory and consequently the size of the Spemann Organizer. The results presented in this manuscript provide evidence that the evolutionarily conserved transcription factor Barhl2 limits the development of the Spemann organizer. In this territory Barhl2 inhibits Wnt activity via its interaction with the co-repressor Groucho and the transcription factor Tcf. It participates to the recruitment of the chromatin remodeling enzyme, Hdac1 that represses the expression of Spemann organizer genes. Using a Xenopus tropicalis Tcf reporter line we demonstrate that Barhl2 inhibitory effect on Groucho-Tcf activities is maintained during embryogenesis and plays a role in the confinement of neural progenitors in the brain. Together, our results provide a new and important mechanism for the control of Wnt transcriptional activity
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Alsaedi, Manal. "The role of WNT inhibitory factor I in adipose tissue development." Thesis, Tennessee State University, 2016. http://pqdtopen.proquest.com/#viewpdf?dispub=10158616.
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Fat tissue is involved in many aspects of biology such as appetite regulation, vascular diseases, diabetes, hypertension, and obesity. It plays an important role in these processes through its endocrine factors and other secretory products. Thus, there is a need to understand better the mechanisms and molecules that control the formation of adipocytes and the expansion of adipose tissue. WNT signaling is one of several important factors that plays a crucial role in development, and may also be important for adipogenesis. The activity of WNT signaling is modulated by a plethora of extracellular modulators that mostly antagonize WNT signaling. The extracellular WNT antagonists consist of four conserved families: Wnt-inhibitory factor 1 (WIF1), secreted frizzled related protein (SFRP), Cerberus, and Dickkopf (Dkk). It has been found that WIF1 is upregulated in abdominal fat tissue in chickens during early development. Thus, we hypothesize that WIF1 plays a role in adipose tissue development by inhibiting WNT signaling, and thereby stimulating adipogenic gene expression. The objective of this research is to examine the in vitro regulation of adipogenesis by WIF1. Mouse WIF1 expression vector (pCMV6-ENTRY-WIF1) was prepared, and used to transfect the mouse pre-adipocyte cell line 3T3-L1.The mRNA levels for WIF1, PPARγ and C/EBPα were then examined with real time RT-qPCR. Results indicate that the transgene was expressed in the transfected cells within 30 hours after transfection, and the mRNA level of WNT target genes and CEBPα were affected. However, the mRNA level of PPARγ was not affected. In conclusion, exogenous WIF1 was expressed in 3T3-L1 cells, at least at the mRNA level. The exogenous WIF1 expression caused an elevation of CEBPα mRNA. Future studies should examine other genes, and more investigation should take place to better understand the mechanisms of adipogenesis.
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Watson, A. T. "ARID1a is an inhibitor of Wnt signalling in Xenopus and human." Thesis, University College London (University of London), 2015. http://discovery.ucl.ac.uk/1468997/.
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Wnt signalling regulates a wide range of events throughout embryonic development, adult homeostasis and the onset of disease. Within the frog Xenopus laevis activation of the Wnt pathway results in one of the first major events during embryogenesis: the establishment of the dorsal-ventral axis. At the molecular level the Wnt pathway in Xenopus embryos is similar to that in many other organisms – including humans – therefore what is learned about protein functions from studies in Xenopus can be extrapolated to other species. My PhD has focussed on the role of ARID1a in the regulation of the Wnt pathway. ARID1a is the largest subunit of the chromatin remodelling BAF complex, which is the vertebrate homologue of the yeast SWI/SNF complex. The BAF complex functions by repositioning nucleosomes within chromatin, and plays a role in a variety of cellular functions such as the regulation of transcription, RNA splicing and DNA damage repair. ARID1a was originally identified in the fruit fly Drosophila, where it was implicated as a repressor of Wg. My studies verify and expand upon this work, investigating the role of ARID1a as a repressor of the Wnt pathway in Xenopus and in Human Embryonic Kidney 293 (HEK293) cells. I carried out a mass spectrometry screen in HEK293 cells and identified two interacting proteins for further study: BCL7a and DDX5. I demonstrate that these two proteins are also able to inhibit Wnt signalling in Xenopus, and suggest mechanisms by which this repression may occur.
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Florczak, Kaya. "Wnt/β-Catenin Signalling Inhibits T-Type Calcium Channels in Cardiomyocytes." Thesis, Université d'Ottawa / University of Ottawa, 2021. http://hdl.handle.net/10393/41983.
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Background: The Wnt/β-catenin signalling pathway is activated in arrhythmogenic heart diseases such as myocardial infarction and heart failure, but it is unclear if the pathway regulates cardiac ion channels and thus may play a role in arrhythmogenesis. Previous PCR array screening from our lab showed that the transcript level of the T-type calcium channel gene Cacna1g was reduced in primary culture of neonatal rat ventricular myocytes (NRVMs) after activation of Wnt/β-catenin signalling with Wnt3a protein (100 ng/ml) or a small molecule activator of the pathway, CHIR (3 µM) (n=3, p<0.01). In this study, we examined the effects of Wnt/β-catenin signalling on T-type calcium channels (Caᴠ3.1), which play a key role in the pacemaker function of the sinoatrial node (SAN). Results: RT-qPCR and western blot demonstrated dose-dependent reductions in Cacna1g mRNA (n=7, p<0.01) and Cav3.1 protein (n=4, p<0.01) in NRVMs after treatment with CHIR (3 μM). There was also a decrease in Cacna1g mRNA in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) after treatment with CHIR (5 μM) (n=4; p<0.001). Patch-clamp recording demonstrated reduced T-type calcium current (ICa,T) in NRVMs after Wnt3a treatment (3 μg/ml) (n=5, p<0.05). In isolated mouse SAN tissue, perfusion with an ICa,T blocker, ML-218 (30 µM), led to dose-dependent reductions in spontaneous beating rate (n=4, p<0.0001) indicating a critical role of ICa,T in SAN pacemaking. In adult rats, activation of Wnt/β-catenin signalling through the application of CHIR in a poloxamer gel to the SAN region did not alter the in vivo heart rate in electrocardiogram (ECG) (n=8, p=0.12). However, ex vivo culture of SAN tissue from the in vivo experiments revealed a reduction intrinsic beating rate in the CHIR treated group (n=7) compared to the control (DMSO) (n=8) (p<0.05). Summary: Wnt/β-catenin signalling inhibits T-type Ca²⁺ current in cardiomyocytes by, at least partly, reduced Cacna1g mRNA and Cav3.1 protein. Activation of Wnt/β-catenin signalling reduces the intrinsic heart rate likely by inhibition of T-type Ca²⁺ current in SAN pacemaker cells.
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Barkell, Alice Mary. "Structural and functional characterisation of Dickkopf4 (Dkk4), a key inhibitor of Wnt signalling." Thesis, University of Leicester, 2013. http://hdl.handle.net/2381/36938.
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A growing weight of experimental evidence indicates that all four secreted protein members of the cysteine-rich Dkk family, Dkk1, Dkk2, Dkk3 and Dkk4, can act as inhibitors of the Wnt pathway. To date, no structural and little functional information has been reported for Dkk4, which has been the focus of this thesis. Full length human Dkk4, including a C-terminal His6 tag, was successfully expressed in E.coli. An effective refolding and purification protocol was developed giving a final yield of 3.0 – 5.0 mg/L of homogenous, correctly folded protein. The activity of Dkk4 was assessed by investigating both its ability to bind the Wnt co-receptor LRP6, and to inhibit Wnt signalling using a variety of experiments including immunoprecipitation, FACS based cell surface binding and Wnt activated luciferase activity. Refolded Dkk4 showed well-dispersed 3D NMR spectra, which allowed the essentially complete, sequence specific assignment of the backbone atoms. The NMR data clearly indicated that both cysteine-rich domains adopt folded structures and strongly suggests a well-defined interface between the two. In addition, a novel interaction between Dkk4 and heparin was demonstrated using chemical shift perturbation experiments. The generation of models of the N-terminal region and C-terminal domain of Dkk4 allowed the perturbation data to be mapped, and revealed that a conformational change is likely to occur upon heparin binding. It is proposed that cell membrane proteins displaying heparan sulphate, a closely related molecule to heparin, may sequester Dkk4 yielding a high local concentration at the cell surface in order the control the Wnt cascade. Interestingly, Dkk4 may be able to interact with both heparin and LRP6 simultaneously rather than competitively.
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Cook, David. "Modulation of canonical Wnt signalling in mesenchymal stem cells using a GSK3beta inhibitor." Thesis, University of York, 2013. http://etheses.whiterose.ac.uk/4609/.
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Multipotent stromal cells/mesenchymal stem cells (MSCs) can differentiate into multiple lineages including osteogenic and adipogenic cells. Wnt signalling has been implicated in controlling MSC fate, but the mechanism is unclear and apparently conflicting data exists. Here I show that a glycogen synthase kinase 3β inhibitor, AR28, is a potent activator of canonical Wnt signalling using β-catenin translocation studies and TCF-reporter assays. AR28 induced axis duplication and secondary regions of chordin expression in Xenopus laevis embryos, when injected into the ventral marginal zone, indicative of canonical Wnt signalling. When human MSCs were grown under adipogenic conditions, AR28 caused a significant dose-dependent reduction in FABP5/BODIPY double-positive cells with a corresponding rescue of proliferation. In assays to determine the effects of AR28 on MSC osteogenesis using standard differentiation inducers (β-glycerophosphate, L-ascorbic acid and dexamethasone), AR28 caused a significant decrease in alkaline phosphatase (ALP) activity compared to vehicle controls, indicative of a reduced osteogenic response. However, when using mild osteogenic stimulation, excluding dexamethasone, increases in both ALP and Alizarin Red mineral staining were identified following AR28 treatment, with corresponding increases in proliferation and cell number. This AR28-induced osteogenic response was blocked by mitomycin C, identifying cell proliferation as an important step in Wnt-induced osteogenesis under these conditions. Pre-treatment of MSCs with AR28 for 7 days before osteogenic induction also increased ALP activity and mineralisation. BMP2 treatment of MSCs was capable of inducing both osteogenic and chondrogenic differentiation, to which AR28 caused a switch towards the osteogenic lineage, with synergistic increases in ALP. AR28 simultaneously caused a decrease in the chondrogenic differentiation of MSCs treated with BMP2 through the down regulation of Sox9 transcription. Together these results highlight the potential of GSK3β inhibitors as therapeutic modulators of canonical Wnt signalling, and there use to treat a multitude of bone related disorders.
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Alamri, Mubarak. "Discovery of WNK-SPAK/OSR1 signalling inhibitors as potential therapeutics." Thesis, University of Birmingham, 2018. http://etheses.bham.ac.uk//id/eprint/8559/.
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Protein kinases are major drug targets for many diseases. Among these are the STE20/SPS1-related proline/alanine-rich kinase (SPAK) and the oxidative- stress-responsive kinase 1 (OSR1), which are two related serine/threonine protein kinases. Both kinases are key components of the WNK-SPAK/OSR1 signalling pathway that has emerged as a key regulator of electrolyte homeostasis, body fluid and blood pressure. Various knock-in and knock-out SPAK and OSR1 mouse models exhibited reduced blood pressure. This highlighted SPAK and OSR1 kinases as promising targets in the treatment of hypertension. Encouraged by this, this project was initiated to discover specific WNK-signalling inhibitors by targeting SPAK and OSR1 kinases as potential novel antihypertensive agents. My work led to the identification of an allosteric pocket located in the highly conserved C-terminal domains of SPAK and OSR1, which influences their kinase activities. Using in silico screening, Rafoxanide, an anti-parasitic agent, was identified as a novel allosteric inhibitor of SPAK and OSR1. Additionally, high throughput screening led to the discovery of the clinically used agent, Verteporfin, as a novel and potent WNK-signalling inhibitor. Moreover, several fragment-binders to the C-terminal domain of OSR1 kinase were identified using NMRfragment based screening. In addition, the NMR backbone assignments of the C-terminal domain of OSR1 kinase were determined and used to map the previously unknown binding site of different OSR1 and SPAK inhibitors. Collectively, these findings have significantly advanced the field of SPAK and OSR1 kinase inhibition and provided key tools that will facilitate the future discovery of other SPAK and OSR1 kinase inhibitors.
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Boral, Debasish. "The Role of SOX2 in Colon Cancer Progression." OpenSIUC, 2014. https://opensiuc.lib.siu.edu/dissertations/911.
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SRY (sex determining region Y)-box 2 (SOX2) is one the embryonic stem cell transcription factors that is capable of reprogramming adult differentiated cells into an induced pluripotent cell. SOX2 is amplified in various types of epithelial cancers and its high its expression correlates with poor prognosis and decreased patient survival. Aberrant Wnt signaling drives the colo-rectal carcinogenic process and is a major determinant of the disease outcome. This study demonstrates that SOX2 counteracts Wnt driven tumor cell proliferation and maintains quiescence in a sub-population of Colo-Rectal Cancer (CRC) cells. High SOX2 expression is found in a sub-group of CRC patients with advanced disease. High SOX2 expression coupled with low Wnt activity was found in SW620 metastatic CRC cell line, while the opposite was true for the isogenic SW480 primary tumor cell line. SOX2 silencing increased Wnt activity and enhanced the oncogenic potential of SW620 cells in vitro and in vivo while over-expression had opposite effects in SW480 cells. SOX2 up-regulates the expression of PTPRK and PHLPP2 protein phosphatase genes which in turn attenuates Wnt activity by interfering with Protein Kinase A, B and C mediated beta catenin phosphorylation at Serine 552 and 675 amino acid residues thereby diminishing its nuclear sequestration and transcriptional activation. Thus SOX2 mitigates growth factor mediated Wnt activation in CRC cells and inhibits cellular proliferation so that these cells are forced to change their oncogene addiction. In effect, high SOX2 expression causes clonal evolution of APC mutant CRC cells from a state of high Wnt dependency to a state of low Wnt dependency in the process making such cells resistant to Wnt inhibitor therapy. Enhanced SOX2 transcriptional activity was associated with increased proportion of cancer cells in G0-G1 phase of cell cycle. Changing SOX2 protein levels in cells had a direct correlation with mRNA levels of RBL2-HUMAN and CDKN2B genes, which serve as regulators of G0 and G1 respectively. SOX2 was shown to physically bind and to the promoter region of these two genes and enhance their transcription. Thus high SOX2 expression, up-regulates the expression of key cell cycle inhibitor genes like RBL2 and CDKN2B and keeps cells in a dormant state. This phenomenon allows colon cancer cells to escape from cytotoxic drug therapy directed at rapidly dividing cells and cause treatment failure and disease relapse.
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Rudge, Felicity. "Genome-wide cDNA and RNAi screening to identify modulators of responses to a novel Wnt signalling inhibitor." Thesis, Cardiff University, 2013. http://orca.cf.ac.uk/58589/.
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Wnt/β-catenin signalling plays a central role in the regulation of multicelluar organism development and in the maintenance of tissue homeostasis in adults. Dysregulation of Wnt signalling resulting in aberrant pathway activation is a key initiating step in the development of a diverse range of cancers, including colorectal cancer, and as such is an important target for therapeutic intervention. A novel Wnt pathway inhibitor, ‘MSC’, has been identified as blocking activated Wnt signalling, specifically through inhibiting the ability of CDK8 and CDK19 to activate nuclear β-catenin/TCF-dependent transcription. However, despite potently inhibiting Wnt-dependent transcription, the ability of MSC to reduce cellular viability was limited. This study aimed to determine genes that whose loss operated with MSC to reduce cell survival. A whole-genome RNAi chemical sensitisation screen identified 3 genes whose depletion in combination with MSC treatment conditionally reduced the viability of HCT116 cells in vitro. The outstanding hit of this screen was Histidyl Aminoacyl tRNA Synthetase (HARS). The identification of this enzyme as an MSC ‘interactor’ suggested links between Wnt signalling and the regulation of translation. BRAF and MED11 RNAi also conferred conditional sensitivity to MSC. Interestingly, MED11 is a component of the Mediator complex, a multiprotein transcription regulatory complex in which CDK8 functions to regulate β-catenin/TCF-dependent transcription, suggesting that mediator complex may be a key target of MSC action. A parallel overexpression screen was initiated to identify novel Wnt pathway activators, and subsequently used to map MSC resistance. Expression of the transcription factors GBX1 and HMGB2, determined to be novel regulators of TCF-dependent transcription, blocked MSC-mediated disruption of Wnt signalling. Overexpression of either gene in a clinical context might therefore be regarded as a contra-indication for MSC-class therapies. These studies have highlighted potential avenues for broadening the scope of MSC activity through the determination of survival and resistance mechanisms, thus the rational design of MSC-combination therapies could be of huge clinical benefit for the treatment of colorectal cancer.
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Bisson, Sarah-Kim, and Sarah-Kim Bisson. "Les inhibiteurs de la voie Wnt dans un modèle animal d'insuffisance rénale chronique avec calcification vasculaire." Master's thesis, Université Laval, 2020. http://hdl.handle.net/20.500.11794/38092.
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En insuffisance rénale chronique (IRC), on observe un déséquilibre minéral qui est associé au développement d’un remodelage osseux anormal et de calcification vasculaire, un processus qui est semblable à la formation osseuse car il implique une trans-différenciation de cellules musculaires lisses vasculaires en cellules ostéoblastiques semblables à celles retrouvées dans l’os. Un lien a été rapporté entre le remodelage osseux ralenti et la calcification vasculaire en IRC mais la cause de ce lien demeure mal comprise. Nous avons étudié l’implication des inhibiteurs de la voie Wnt dans ce lien dans un modèle d’IRC chez le rat avec calcification vasculaire induite par un supplément de calcium, phosphore et vitamine D (Ca/P/vitD). Les animaux IRC+Ca/P/vitD présentaient de la calcification vasculaire, un remodelage osseux ralenti, un défaut de minéralisation et des niveaux sériques et vasculaires d’inhibiteurs de la voie Wnt élevés. La présence dans le vaisseau calcifié d’un inhibiteur de la voie Wnt de source ostéocytaire, la sclérostine, suggère que les cellules ostéoblastiques acquièrent un phénotype ostéocytaire. De plus, les inhibiteurs de la voie Wnt produits par le vaisseau pourraient avoir des conséquences sur l’os, ce qui cadrerait avec la formation ralentie et le défaut de minéralisation observés. Les inhibiteurs de la voie Wnt en circulation pourraient aussi avoir des effets vasculaires puisque les vaisseaux calcifiés présentaient des signes d’inhibition de la voie Wnt/β-caténine, les niveaux d’inhibiteurs de la voie Wnt ne semblant toutefois pas associés à un ralentissement de la calcification. Nos résultats mettent donc en lumière une implication potentielle des inhibiteurs de la voie Wnt dans le lien entre l’os et le vaisseau en IRC.En insuffisance rénale chronique (IRC), on observe un déséquilibre minéral qui est associé au développement d’un remodelage osseux anormal et de calcification vasculaire, un processus qui est semblable à la formation osseuse car il implique une trans-différenciation de cellules musculaires lisses vasculaires en cellules ostéoblastiques semblables à celles retrouvées dans l’os. Un lien a été rapporté entre le remodelage osseux ralenti et la calcification vasculaire en IRC mais la cause de ce lien demeure mal comprise. Nous avons étudié l’implication des inhibiteurs de la voie Wnt dans ce lien dans un modèle d’IRC chez le rat avec calcification vasculaire induite par un supplément de calcium, phosphore et vitamine D (Ca/P/vitD). Les animaux IRC+Ca/P/vitD présentaient de la calcification vasculaire, un remodelage osseux ralenti, un défaut de minéralisation et des niveaux sériques et vasculaires d’inhibiteurs de la voie Wnt élevés. La présence dans le vaisseau calcifié d’un inhibiteur de la voie Wnt de source ostéocytaire, la sclérostine, suggère que les cellules ostéoblastiques acquièrent un phénotype ostéocytaire. De plus, les inhibiteurs de la voie Wnt produits par le vaisseau pourraient avoir des conséquences sur l’os, ce qui cadrerait avec la formation ralentie et le défaut de minéralisation observés. Les inhibiteurs de la voie Wnt en circulation pourraient aussi avoir des effets vasculaires puisque les vaisseaux calcifiés présentaient des signes d’inhibition de la voie Wnt/β-caténine, les niveaux d’inhibiteurs de la voie Wnt ne semblant toutefois pas associés à un ralentissement de la calcification. Nos résultats mettent donc en lumière une implication potentielle des inhibiteurs de la voie Wnt dans le lien entre l’os et le vaisseau en IRC.
Chronic kidney disease (CKD) patients suffer from a dysregulation of minerals levels which is associated with abnormal bone remodeling and vascular calcification, a process similar to bone formation in that it implies the trans-differentiation of vascular smooth muscle cells into osteoblast-like cells. A link was reported between decreased bone formation and vascular calcification in CKD, but the cause of this link remains unclear. We have studied the involvement of Wnt pathway inhibitors in this process in a rat model of CKD with vascular calcification induced by a calcium, phosphorus and vitamin D supplement (Ca/P/vitD). CKD+Ca/P/vitD rats presented with vascular calcification, decreased bone turnover, defective mineralization and increased levels of circulating and vascular Wnt inhibitors. The expression by the calcified vessel of sclerostin, a Wnt inhibitor typically produced by osteocytes, suggests that vascular osteoblast-like cells could acquire an osteocytic phenotype as osteoblasts do in bone. Moreover, vascular Wnt inhibitors could have consequences on bone and contribute to the decrease in bone formation and the mineralization defect. The high circulating levels of Wnt inhibitors could also have vascular effects, which is supported by the fact that the Wnt/β-catenin pathway appears to be inhibited in the calcified vessels despite the fact that high levels of Wnt inhibitors were not correlated with a decrease in the severity of vascular calcification. Our results therefore suggest an implication of Wnt pathway inhibitors in the bone-vessels link that is frequently observed in CKD.
Chronic kidney disease (CKD) patients suffer from a dysregulation of minerals levels which is associated with abnormal bone remodeling and vascular calcification, a process similar to bone formation in that it implies the trans-differentiation of vascular smooth muscle cells into osteoblast-like cells. A link was reported between decreased bone formation and vascular calcification in CKD, but the cause of this link remains unclear. We have studied the involvement of Wnt pathway inhibitors in this process in a rat model of CKD with vascular calcification induced by a calcium, phosphorus and vitamin D supplement (Ca/P/vitD). CKD+Ca/P/vitD rats presented with vascular calcification, decreased bone turnover, defective mineralization and increased levels of circulating and vascular Wnt inhibitors. The expression by the calcified vessel of sclerostin, a Wnt inhibitor typically produced by osteocytes, suggests that vascular osteoblast-like cells could acquire an osteocytic phenotype as osteoblasts do in bone. Moreover, vascular Wnt inhibitors could have consequences on bone and contribute to the decrease in bone formation and the mineralization defect. The high circulating levels of Wnt inhibitors could also have vascular effects, which is supported by the fact that the Wnt/β-catenin pathway appears to be inhibited in the calcified vessels despite the fact that high levels of Wnt inhibitors were not correlated with a decrease in the severity of vascular calcification. Our results therefore suggest an implication of Wnt pathway inhibitors in the bone-vessels link that is frequently observed in CKD.
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Browne, Andrew. "The effects and regulation of the Wnt inhibitor Dickkopf-1 and the mechanistic target of rapamycin in osteotropic cancers." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2017. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-228981.
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As solid tumor types, breast and prostate cancer are rivalled only by lung cancer in their propensity to metastasize to bone in the later stages of disease. At advanced stages of disease, approximately 80% of breast and 90% of prostate cancer patients will present with bone metastases. Bone metastases are often a painful conclusion to the lives of these patients, resulting in bone pain, hypercalcemia, pathological fractures and spinal cord compression. The culmination of these comorbidities considerably reduces a patient’s quality of life and prolonged survival. Hormone depletion is used as a first line of treatment in the majority of cases, negatively regulating bone health due to increased bone resorption by osteoclasts and decreased bone formation by osteoblasts. Not only is bone integrity undermined, but this action of increased bone turnover is beneficial for the colonization of metastasizing cells which co-opt and enhance the same mechanisms to establish and maintain their own growth. This is termed ‘the vicious cycle’ of osteolytic bone metastasis.
Current research approaches aim to identify bone-targeted therapies which not only inhibit tumor growth but concurrently protect bone. In this study, Dickkopf-1 (DKK-1), mechanistic target of rapamycin (mTOR) and p38 mitogen-activated kinases (p38 MAPK) are presented as novel targets. Pro-tumor roles have been described for all and clinical trials are currently investigating their efficacy in different cancer types. In normal bone biology DKK-1 is an inhibitor of the canonical Wnt signaling pathway which promotes osteoblastogenesis while mTOR signaling is a promoter of osteoclastogenesis. P38 MAPK inhibitors have been shown to regulate DKK-1 expression and bone destruction in preclinical models of multiple myeloma. The aims of this current study were to 1) investigate the role of DKK-1 in the biology of osteotropic breast cancer, 2) to assess the potential bone protective effects of mTOR inhibition by everolimus in the context of osteotropic cancers and 3) to test the hypothesis that p38 MAPK is a regulator of DKK-1 expression in prostate cancer, potentially supporting an osteolytic phenotype by impairing osteoblastogenesis.
In aim 1, analysis of a breast cancer tissue microarray demonstrated that DKK-1 expression was elevated in advanced and invasive tumor stages. Strikingly, positive DKK-1 expression correlated with a significantly reduced survival rate only in estrogen receptor-negative (ER-) breast cancer patients compared to patients with tumors which were negative for DKK-1 expression. In MDA-MB-231 breast cancer xenograft models, neutralization of secreted DKK-1 by treating mice with the monoclonal DKK-1 antibody BHQ880 or knocking out the expression of DKK-1 in MDA-MB-231 cells using CRISPR-Cas9 mediated gene editing, resulted in reduced tumor growth and burden by ≥ 50% (p < 0.05). In aim 2, the mTOR inhibitor everolimus is presented as an anti-tumor and bone-protective agent. The anti-tumor effects of everolimus were confirmed in two subcutaneous tumor models and a model of breast cancer bone metastasis, were tumor burden in the bone was reduced by 45.4% (p < 0.01). Bone loss induced by a hormone-deprived environment in ovariectomized mice was prevented with everolimus treatment as was bone destruction in the metastasis model. In more detail, it could be shown that everolimus maintained osteoblast function while specifically inhibiting osteoclast function. In aim 3, p38 MAPK is presented as a regulator of DKK-1 in prostate cancer. While the activation of p38 MAPK upregulated DKK-1, inhibition of p38 MAPK using small molecule inhibitors and siRNAs inhibited DKK-1 expression. Furthermore, assessment of different p38 MAPK isoforms revealed MAPK11 as the most effective regulator of DKK-1 and inhibition of DKK-1 by interfering with p38 MAPK signaling was sufficient to prevent the inhibitory effects of prostate cancer-derived DKK-1 on osteoblastogenesis in vitro.
This study has assessed multiple targets and their concurrent roles in cancer and bone cell biology. Specifically, DKK-1 has been proven to be a tumor promoter in ER- breast cancer and can be targeted therapeutically to inhibit tumor growth. MTOR inhibition by everolimus has been shown to be an effective mono-therapy in ER- breast cancer, inhibiting the growth of subcutaneous tumor and bone metastases and preventing bone loss induced by estrogen ablation. This further supports its use in postmenopausal women with breast cancer who are predisposed to developing osteoporosis and bone metastases. It also supports the use of everolimus in hormone receptor-negative or triple receptor-negative breast cancer, for which it has not yet been approved. A clear link has been made between p38 MAPK signaling and DKK-1 expression in prostate cancer and its consequent regulation of osteoblastogenesis. A future focus on the inhibition of a specific MAPK isoform, MAPK11 in particular, may help in translating these encouraging in vitro results into promising pre-clinical trials in vivo.
As a whole, these investigations provide a foundation for further research and could be valuable for the design of future clinical trials, leading to improvements in the treatment and prognosis of osteolytic bone metastases.
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Yao, Hisayuki. "AV-65, a novel Wnt/β-catenin signal inhibitor, successfully suppresses progression of multiple myeloma in a mouse model." Kyoto University, 2012. http://hdl.handle.net/2433/157443.
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Funke, Robin [Verfasser], and Jörg [Akademischer Betreuer] Distler. "Epigenetisches Silencing von endogenen Inhibitoren des WNT-Signalweges durch Promoter Methylierung in der Systemischen Sklerose / Robin Funke. Betreuer: Jörg Distler." Erlangen : Universitätsbibliothek der Universität Erlangen-Nürnberg, 2012. http://d-nb.info/1024198901/34.
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Matzelle, Melissa M. "Inflammation Inhibits Osteoblast-Mediated Bone Formation in Rheumatoid Arthritis and Regulates the Wnt and BMP Signaling Pathways: A Dissertation." eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/596.
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Osteoclast-mediated focal articular bone erosion is a hallmark of rheumatoid arthritis, a disease of inflammation-induced bone loss. Inflammation in the bone microenvironment enhances osteoclast differentiation leading to bone erosion. Simultaneously, inflammation also inhibits osteoblast-mediated bone formation, further contributing to the net loss of bone. Previous studies have shown a paucity of mature osteoblasts at eroded bone surfaces correlating with suppression of bone formation and upregulation of antagonists of the Wnt pathway, a signaling cascade essential for osteoblast lineage commitment. Despite these observations, the exact pathogenesis of impaired bone formation in the setting of inflammation is not clearly understood.
This dissertation aims to delineate the mechanisms by which inflammation suppresses osteoblast differentiation and activity in inflammatory arthritis. Specifically, this research elucidates how inflammation-induced alterations in the Wnt and bone morphogenetic protein (BMP) osteogenic signaling pathways contribute to bone loss and formation at distinct inflammatory microenvironments within the bone. Secondly, the means by which cellular mediators, including lymphocytes and macrophages, facilitate bone erosion and formation was addressed.
Taken together, the research in this dissertation underscores the relationship between inflammation-induced bone loss and alterations in osteogenic signaling. Using an innovative murine inflammatory arthritis model, this study definitively demonstrates that resolving inflammation promotes osteoblast-mediated bone formation. Repair of erosions correlates with upregulation of synovial expression of Wnt10b, a Wnt agonist, and downregulation of sFRP1 and sFRP2, Wnt antagonists. This work also directly evaluates the contribution of sFRP1 to inflammation-induced bone destruction. Furthermore, this research demonstrates that expression of BMP3, a negative regulator of BMP signaling, is upregulated in osteoblasts by IL-17, a pro-inflammatory cytokine. BMP3-expressing osteoblasts are also observed at erosion sites in murine arthritis. Lastly, evaluation of the mediators of inflammation-induced periosteal bone formation implicates BMP2 as a means by which inflammation may positively regulate osteoblast function.
This dissertation further elucidates the role of T cells and macrophages in the erosion and formation processes, respectively. In the absence of lymphocytes, bone erosion occurred normally, demonstrating that RANKL-expressing lymphocytes are not absolutely required for the bone erosion. Preliminary studies also suggest that M2 macrophages are potential mediators of bone formation via the expression of BMP2.
In conclusion, this dissertation explores the ability of inflammation to act as a rheostat, which controls the fate of bone by modulating not only osteoclast differentiation, but also osteogenic signaling pathways and cellular mediators in the bone microenvironment. The soluble mediators and cell types identified in this research highlight novel mechanisms by which inflammation may regulate osteoblast activity within the bone microenvironment. Collectively, these data imply that strict control of inflammation may be necessary in order to create an anabolic environment that preserves bone architecture in diseases of inflammation-induced bone loss.
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Nagao, Rina. "Growth inhibition of imatinib-resistant CML cells with the T315I mutation and hypoxia-adaptation by AV65 - a novel Wnt/β-catenin signaling inhibitor." Kyoto University, 2012. http://hdl.handle.net/2433/157488.
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Barat, Samarpita [Verfasser], and Ruben [Akademischer Betreuer] Plentz. "Gamma-secretase inhibitor IX (GSI) impairs concomitant activation of Notch and wnt-beta-catenin pathways in CD44+ gastric cancer / Samarpita Barat ; Betreuer: Ruben Plentz." Tübingen : Universitätsbibliothek Tübingen, 2016. http://d-nb.info/1199615560/34.
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Engel, James L. "The Psuedomonas syringae type III effector HopE1 interacts and inhibits an Arabidopsis WNK kinase." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2008. http://wwwlib.umi.com/cr/ucsd/fullcit?p1459876.
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Thesis (M.S.)--University of California, San Diego, 2008.Title from first page of PDF file (viewed December 18, 2008). Available via ProQuest Digital Dissertations. Includes bibliographical references (p. 47-50).
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Sack, Ulrike. "New insights into S100A4-induced colon cancer metastasis." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2011. http://dx.doi.org/10.18452/16313.
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S100A4 spielt eine zentrale Rolle für die Metastasierung des Dickdarmkrebses. Die Hemmung der S100A4 Expression stellt damit einen vielversprechenden therapeutischen Ansatz dar. Die vorliegende Arbeit präsentiert Niklosamid und Calcimycin als neue Inhibitoren der S100A4 Transkription. In Kolonkarzinomzellen, die mit einem der beiden Inhibitoren behandelt wurden, wurde die S100A4 Expression konzentrations- und zeitabhängig unterdrückt. Des Weiteren war die Zellmigration und -invasion in Abhängigkeit von S100A4 in behandelten Zellen vermindert. Niklosamid und Calcimycin Behandlung verhinderten die Zellproliferation und die Koloniebildung von Kolonkarzinomzellen. Beide Inhibitoren hemmten den konstitutiv aktiven Wnt Pathway von Kolonkarzinomzellen. Calcimycin Behandlung verminderte die Expression von beta-catenin. Niklosamid hemmte die Bildung des beta-catenin/TCF Komplexes und unterband damit die Expression von Wnt Pathway Genen, wie z.B. S100A4. Im Rahmen dieser Arbeit wurde ein in vivo Tiermodell entwickelt, mit dem die S100A4-induzierte Metastasierung mit Hilfe von nicht-invasivem Biolumineszenz Imaging visualisiert werden konnte. In diesem Model konnte gezeigt werden, dass Niklosamid signifikant die S100A4 Expression im Tumor vermindert und damit die Metastasierung hemmt. Des Weiteren zeigt diese Arbeit, dass S100A4 die Expression des Wnt Pathway Antagonisten DKK-1 in Kolonkarzinomzellen hemmt. DKK-1 selbst konnte als endogener Inhibitor der S100A4 Expression identifiziert werden. Zusammenfassend beschreibt die vorliegende Arbeit einen neuen regulativen Mechanismus im Wnt Pathway, der die S100A4 Expression im Kolonkarzinom fördert. Diese Beobachtung verdeutlicht die Notwendigkeit für wirksame S100A4 Inhibitoren, wie Niklosamid und Calcimycin, die das Potenzial haben, in einer klinischen Anwendung die Metastasierung von Kolonkarzinompatienten mit erhöhter S100A4 Expression zu hemmen und damit deren Überlebenschance wesentlich zu verbessern.S100A4 promotes metastasis in colon cancer patients thereby reducing their five-year survival chances to less than 10%. Consequently, inhibition of S100A4 expression is a promising strategy for anti-metastatic treatment of colon cancer patients. The present study characterizes the small molecules niclosamide and calcimycin as transcriptional inhibitors of S100A4 which reduced S100A4 expression concentration- and time-dependently. Niclosamide and calcimycin treatment restricted cell migration, invasion and wound healing capabilities in a S100A4-specific manner, and inhibited cell proliferation and colony formation of colon cancer cells. Both small molecule inhibitors interfere with the constitutively active Wnt pathway. Targeting β-catenin expression by calcimycin or interfering with the β-catenin/TCF transcription activating complex by niclosamide resulted in reduced Wnt target gene transcription, among them S100A4. The study further presents a human colon cancer xenograft mouse model for monitoring S100A4-induced metastasis formation via non-invasive bioluminescence imaging. Treatment of xenograft mice with niclosamide resulted in a significant reduction of the S100A4 mRNA level in the tumor accompanied by inhibition of metastasis formation. Moreover, this study presents evidence that S100A4 is an inhibitor of DKK-1 expression. In colon cancer cells DKK-1 and S100A4 expression was negatively correlated. Ectopic S100A4 overexpression inhibited DKK-1 expression. Targeting S100A4 via shRNA recovered the repressed DKK-1 expression and vice versa. In summary, the study describes a novel positive feedback loop in the Wnt pathway regulation formed by S100A4 repressing its antagonist DKK-1. This novel mechanism further strengthens the need for S100A4 inhibitors such as niclosamide or calcimycin. Consequently, such small molecules provide immense potential for the treatment of colon cancer patients who are at high risk for S100A4-induced colon cancer metastasis.
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Salvagnini, Claudio. "Thrombin inhibitors grafting on polyester membranes for the preparation of blood-compatible materials." Université catholique de Louvain, 2005. http://edoc.bib.ucl.ac.be:81/ETD-db/collection/available/BelnUcetd-11232005-103604/.
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The design of biomaterials, historically initiated and developed by physicians and engineers, in the last decades has slowly shifted toward a more biochemical based approach. For the replacement, repair and regeneration of tissues scientists are now focusing on materials that stimulate specific biological response at the molecular level. These biomaterials have already shown interesting applications in cell proliferation, differentiation, and extracellular matrix production and organization when the material modifications are designed to elicit specific interactions with cell integrins. In the present work we propose the application of this strategy for the development of blood-compatible materials. We first identified, in the coagulation cascade a key enzyme that constitute a valuable biological target for the development of anti-thrombogenic compounds. Piperazinyl-amide derivatives of N-alfa-(3-trifluoromethyl-benzenesulfonyl)-L-arginine were synthesized as graftable thrombin inhibitors. These inhibitors provided a spacer arm for surface grafting and a fluorine tag for XPS (X-ray photoelectron spectroscopy) detection. The possible disturbance of biological activity due to a variable spacer-arm fixed on the N-4 piperazinyl position was evaluated in vitro against human alfa-thrombin, in silico by molecular modelling and via X-ray diffraction study. Selected inhibitors, having inhibition potency in the mM range, were grafted on polyesters surface via wet chemistry and photochemical activation treatments. Wet chemistry surface grafting was performed by specific hydroxyl chain-ends activation and resulted in bioactive molecules fixation of 20-300pmol/cm2. The photochemical grafting was performed using a molecular clip providing an aromatic azide, for nitrene insertion into a polymer, and an activated ester for grafting of tag compounds. This grafting technique resulted in a dramatic increase in fixed bioactive signals (up to nmol/cm2). The material blood-compatibilization induced by the surface fixation of the inhibitors, was measured by a static blood clot weight measurement test. The wet chemistry grafting technique resulted in moderate blood-compatibilization while by the photochemical grafting method important decrease in surface blood clot formation was observed. In the latter case, the blood response to material contact was found to be strongly affected by the polyester surface photo-degradation induced by the activation treatment.
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Benchekroun, Mohamed. "Synthèse multicomposants et évaluation pharmacologique de nouveaux adduits de Ugi et de Passerini pour le traitement de la maladie d'Alzheimer." Thesis, Besançon, 2014. http://www.theses.fr/2014BESA3007/document.
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La maladie d'Alzheimer est la pathologie neurodégénérative la plus courante affectant les personnes âgées. Cette neuropathologie se caractérise par une étiologie complexe dont le déficit en acétylcholine, les plaques amyloïdes, les dégénérescences neurofibrillaires ou le stress oxydant en sont les principaux acteurs.Au cours de cette thèse, nous nous sommes intéressés à l'application des réactions multicomposants de Ugi et de Passerini, pour la synthèse de nouveaux adduits multi-cibles basées sur différents motifs antioxydants et anticholinestérasiques. Ces réactions permettent d'accéder à une vaste diversité chimique en une étape, ce qui les rend adaptées pour la synthèse rapide de molécules ayant plusieurs pharmacophores d'intérêt et ciblant ainsi différentes cause étiologiques de la maladie d'Alzheimer.Au total, 56 composés finaux, répartis dans cinq séries, ont été synthétisés :les alpha-acylaminocarboxamides prototypes (série A)les hybrides tacrine-acide férulique (série B)les hétérotrimères tacrine-mélatonine-acides antioxydant (série C)> les hybrides donépézil-acide férulique (série D)^ les dérivés chromone (série E)Toutes les séries ont été évaluées pour leur capacité à inhiber les enzymes cholinestérases et leur pouvoir antioxydant. L'hépatotoxicité des séries B et C. portant un motif tacrine, a été évaluée sur les cellules HepG2. Par ailleurs, l'étude de la série B a été complétée par d'autres tests pharmacologiques, physico-chimiques et toxicologiques.Ces différents travaux démontrent et valident l'utilisation des réactions de Ugi et de Passerini dans le développement de molécules multi-cibles pour le traitement potentiel de la maladie d'AlzheimerAlzheimer's disease (AD) is thé most common type of dementia affecting elderly people. This neuropathology is characterized by a highiy complex and intricated etiology including cholinergic déficit, amyloid deposits, neurofibrillary tangles and oxidative stress.During this thesis, we sought to apply Ugi and Passerini multicomponent reactions for thé synthesis of new multi-target adducts based on différent antioxidant and anticholinergic scaffolds.Thèse réactions provides access to a broad range of chemical diversity in a one-pot fashion, which makes them suitable for thé expeditious synthesis of molécules having several pharmacophores of interest and hitting différent targets related to thé multifaceted etiology of Alzheimer's disease.A total of 56 final compounds, spread over 5 séries, hâve been synthesized:alpha-acylaminocarboxamides prototypes (A séries)tacrine-ferulic acid hybrids (B séries)tacrine-melatonin-antioxydant acids heterotrimers (C séries)donepezil-ferulic acid hybrids (D séries)Chromone derivatives (E séries)Ail thé séries were tested for their ability to inhibit thé cholinesterases enzymes and for their antioxidant power. Hepatotoxicity of thé B and C séries, bearing a tacrine fragment, was evaluated on HepG2 cells. Moreover, thé study of thé B séries was supplemented by further pharmacological, physicochemical and toxicological tests (NMR conformational study, neuroprotection on SH-SY5Y cells. self-induced Abetai.42 peptide aggregation inhibition, docking ADMET).Such work demonstrated and validated thé use of Ugi and Passerini reactions for thé development of new multi-target directed molécules for thé potential treatment of AD
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Morales, Espejo Jesus Hector. "Atmospheric corrosion of quaternary bronzes (Cu-Sn-Zn-Pb): laboratory tests (accelerated ageing in wet & dry conditions)and field studies (the Bottego monument in Parma, Italy)." Master's thesis, Alma Mater Studiorum - Università di Bologna, 2011. http://amslaurea.unibo.it/2386/.
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The interactions between outdoor bronzes and the environment, which lead to bronze corrosion, require a better understanding in order to design effective conservation strategies in the Cultural Heritage field. In the present work, investigations on real patinas of the outdoor monument to Vittorio Bottego (Parma, Italy) and laboratory studies on accelerated corrosion testing of inhibited (by silane-based films, with and without ceria nanoparticles) and non-inhibited quaternary bronzes are reported and discussed. In particular, a wet&dry ageing method was used both for testing the efficiency of the inhibitor and for patinating bronze coupons before applying the inhibitor. A wide range of spectroscopic techniques has been used, for characterizing the core metal (SEM+EDS, XRF, AAS), the corroded surfaces (SEM+EDS, portable XRF, micro-Raman, ATR-IR, Py-GC-MS) and the ageing solutions (AAS).
The main conclusions were:
1. The investigations on the Bottego monument confirmed the differentiation of the corrosion products as a function of the exposure geometry, already observed in previous works, further highlighting the need to take into account the different surface features when selecting conservation procedures such as the application of inhibitors (i.e. the relative Sn enrichment in unsheltered areas requires inhibitors which effectively interact not only with Cu but also with Sn).
2. The ageing (pre-patination) cycle on coupons was able to reproduce the relative Sn enrichment that actually happens in real patinated surfaces, making the bronze specimens representative of the real support for bronze inhibitors.
3. The non-toxic silane-based inhibitors display a good protective efficiency towards pre-patinated surfaces, differently from other widely used inhibitors such as benzotriazole (BTA) and its derivatives.
4. The 3-mercapto-propyl-trimethoxy-silane (PropS-SH) additivated with CeO2 nanoparticles generally offered a better corrosion protection than PropS-SH.
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Melchert, Juliane [Verfasser], Tomas [Akademischer Betreuer] Pieler, and Ernst A. [Akademischer Betreuer] Wimmer. "Expression screen for Wnt signaling-like phenotypes identifies Fam132b as a novel inhibitor of BMP signaling in Xenopus / Juliane Melchert. Gutachter: Tomas Pieler ; Ernst A. Wimmer. Betreuer: Tomas Pieler." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2014. http://d-nb.info/1048469948/34.
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Browne, Andrew John [Verfasser], Lorenz [Akademischer Betreuer] [Gutachter] Hofbauer, and Martin [Gutachter] Bornhäuser. "The effects and regulation of the Wnt inhibitor Dickkopf-1 and the mechanistic target of rapamycin in osteotropic cancers / Andrew Browne ; Gutachter: Lorenz Hofbauer, Martin Bornhäuser ; Betreuer: Lorenz Hofbauer." Dresden : Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2017. http://d-nb.info/1140735217/34.
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Browne, Andrew [Verfasser], Lorenz [Akademischer Betreuer] [Gutachter] Hofbauer, and Martin [Gutachter] Bornhäuser. "The effects and regulation of the Wnt inhibitor Dickkopf-1 and the mechanistic target of rapamycin in osteotropic cancers / Andrew Browne ; Gutachter: Lorenz Hofbauer, Martin Bornhäuser ; Betreuer: Lorenz Hofbauer." Dresden : Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2017. http://d-nb.info/1140735217/34.
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Lintereur, Phillip. "EFFECTS OF SOURCE WATER BLENDING FOLLOWING TREATMENT WITH SODIUM SILICATE AS A CORROSION INHIBITOR ON METAL RELEASE WITHIN A WAT." Doctoral diss., University of Central Florida, 2008. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/2967.
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A study was conducted to investigate and quantify the effects of corrosion inhibitors on metal release within a pilot distribution system while varying the source water. The pilot distribution system consisted of pre-existing facilities from Taylor et al (2005). Iron, copper, and lead release data were collected during four separate phases of operation. Each phase was characterized by the particular blend ratios used during the study. A blended source water represented a water that had been derived from a consistent proportion of three different source waters. These source waters included (1) surface water treated through enhanced coagulation/sedimentation/filtration, (2) conventionally treated groundwater, and (3) finished surface water treated using reverse osmosis membranes. The corrosion inhibitors used during the study were blended orthophosphate (BOP), orthophosphate (OP), zinc orthophosphate (ZOP), and sodium silicate (Si). This document was intended to cite the findings from the study associated with corrosion treatment using various doses of sodium silicate. The doses were maintained to 3, 6, and 12 mg/L as SiO2 above the blend-dependent background silica concentration. Sources of iron release within the pilot distribution system consisted of, in the following order of entry, (1) lined cast iron, (2) un-lined cast iron, and (3) galvanized steel. Iron release data from these materials was not collected for each individual iron source. Instead, iron release data represented the measurement of iron upon exposure to the pilot distribution system in general. There was little evidence to suggest that iron release was affected by sodium silicate. Statistical modeling of iron release suggested that iron release could be described by the water quality parameters of alkalinity, chlorides, and pH. The R2 statistic implied that the model could account for only 36% of the total variation within the iron release data set (i.e. R2 = 0.36). The model implies that increases in alkalinity and pH would be expected to decrease iron release on average, while an increase in chlorides would increase iron release. The surface composition of cast iron and galvanized steel coupons were analyzed using X-ray photoelectron spectroscopy (XPS). The surface analysis located binding energies consistent with Fe2O3, Fe3O4, and FeOOH for both cast iron and galvanized steel. Elemental scans detected the presence of silicon as amorphous silica; however, there was no significant difference between scans of coupons treated with sodium silicate and coupons simply exposed to the blended source water. The predominant form of zinc found on the galvanized steel coupons was ZnO. Thermodynamic modeling of the galvanized steel system suggested that zinc release was more appropriately described by Zn5(CO3)2(OH)6. The analysis of the copper release data set suggested that treatment with sodium silicate decreased copper release during the study. On average the low, medium, and high doses decreased copper release, when compared to the original blend source water prior to sodium silicate addition, by approximately 20%, 30%, and 50%, respectively. Statistical modeling found that alkalinity, chlorides, pH, and sodium silicate dose were significant variables (R2 = 0.68). The coefficients of the model implied that increases in pH and sodium silicate dose decreased copper release, while increases in alkalinity and chlorides increased copper release. XPS for copper coupons suggested that the scale composition consisted of Cu2O, CuO, and Cu(OH)2 for both the coupons treated with sodium silicate and those exposed to the blended source water. Analysis of the silicon elemental scan detected amorphous silica on 3/5 copper coupons exposed to sodium silicate. Silicon was not detected on any of the 8 control coupons. This suggested that sodium silicate inhibitor varied the surface composition of the copper scale. The XPS results seemed to be validated by the visual differences of the copper coupons exposed to sodium silicate. Copper coupons treated with sodium silicate developed a blue-green scale, while control coupons were reddish-brown. Thermodynamic modeling was unsuccessful in identifying a controlling solid that consisted of a silicate-based cupric solid. Lead release was generally decreased when treated with sodium silicate. Many of the observations were recorded below the detection limit (1 ppb as Pb) of the instrument used to measure the lead concentration of the samples during the study. The frequency of observations below the detection limit tended to increase as the dose of sodium silicate increased. An accurate quantification of the effect of sodium silicate was complicated by the observations recorded below detection limit. If the lead concentration of a sample was below detection limit, then the observation was recorded as 1 ppb. Statistical modeling suggested that temperature, alkalinity, chlorides, pH, and sodium silicate dose were important variables associated with lead release (R2 = 0.60). The exponents of the non-linear model implied that an increase in temperature, alkalinity, and chlorides increased lead release, while an increase in pH and sodium silicate dose were associated with a decrease in lead release. XPS surface characterization of lead coupons indicated the presence of PbO, PbO2, PbCO3, and Pb3(OH)2(CO3)2. XPS also found evidence of silicate scale formation. Thermodynamic modeling did not support the possibility of a silicate-based lead controlling solid. A solubility model assuming Pb3(OH)2(CO3)2 as the controlling solid was used to evaluate lead release data from samples in which lead coupons were incubated for long stagnation times. This thermodynamic model seemed to similarly describe the lead release of samples treated with sodium silicate and samples exposed to the blended source water. The pH of each sample was similar, thus sodium silicate, rather than the corresponding increase in pH, would appear to be responsible if a difference had been observed. During the overall study, the effects of BOP, OP, ZOP, and Si corrosion inhibitors were described by empirical models. Statistically, the model represented the expected value, or mean average, function. If these models are to be used to predict a dose for copper release, then the relationship between the expected value function and the 90th percentile must be approximated. The USEPA Lead and Copper Rule (LCR) regulates total copper release at an action level of 1.3 mg/L. This action level represents a 90th percentile rather than a mean average. Evaluation of the complete copper release data set suggested that the standard deviation was proportional to the mean average of a particular treatment. This relationship was estimated using a linear model. It was found that most of the copper data sub-sets (represented by a given phase, inhibitor, and dose) could be described by a normal distribution. The information obtained from the standard deviation analysis and the normality assumption validated the use of a z-score to relate the empirical models to the estimated 90th percentile observations. Since an analysis of the normality and variance (essentially contains the same information as the standard deviation) are required to assess the assumptions associated with an ANOVA, an ANOVA was performed to directly compare the effects of the inhibitors and corresponding doses. The findings suggested that phosphate-based inhibitors were consistently more effective than sodium silicate when comparing the same treatment levels (i.e. doses). Among the phosphate-based inhibitors, the effectiveness of each respective treatment level was inconsistent (i.e. there was no clear indication that any one phosphate-based inhibitor was more effective than the other). As the doses increased for each inhibitor, the results generally suggested that there was a corresponding tendency for copper release to decrease.Ph.D.
Department of Civil and Environmental Engineering
Engineering and Computer Science
Electrical Engineering PhD
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Martinez, Merizalde Balarezo Nelson, Rivera Mark Monroe, and Romina A. Tejada. "Re: Maud Rijnders, Ronald de Wit, Joost L. Boormans, Martijn P.J. Lolkema, Astrid A.M. van der Veldt. Systematic Review of Immune Checkpoint Inhibition in Urological Cancers. Eur Urol. 2017;72:411–23." Elsevier B.V, 2018. http://hdl.handle.net/10757/624728.
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Balke-Want, Hyatt [Verfasser], Roman [Akademischer Betreuer] Thomas, and Alexander [Akademischer Betreuer] Quaas. "Identifizierung genetischer und funktioneller Mechanismen für Sensitivität und Resistenz des Bronchialkarzinoms gegenüber Tyrosinkinase-Inhibitoren / Hyatt Balke-Want ; Akademische Betreuer: Roman Thomas, Alexander Quaas." Köln : Deutsche Zentralbibliothek für Medizin, 2018. http://d-nb.info/117665344X/34.
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Ganguly, Atish. "Wnt8 Is a Novel Target of the Dorsal/Twist/Snail Network and an Inhibitor of Dorsal in the Gastrulating Drosophila Embryo: A Dissertation." eScholarship@UMMS, 2004. http://escholarship.umassmed.edu/gsbs_diss/201.
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The work in presented in this thesis identifies Drosophila Wnt8 as a novel zygotic target of the Dorsal/Twist/Snail network using a microarray analysis to identify differentially expressed genes in maternal dorsal mutant and gain-of-function Toll10b embryos as compared to wild type. In-situ hybridization with a Wnt8 antisense RNA probe revealed a fairly complex expression pattern in the early embryo. No maternal expression was observed and the first zygotic expression appeared at stage 4 at the poles. This was followed by patchy and relatively weak expression in the presumptive mesoderm with stronger expression in the mesectoderm and later the neuroectoderm. These expression required the Dorsal/Twist/Snail network with some input from Delta in the neuroectoderm. All embryonic Wnt8 expression ceased after late stage 10. Snail was found to repress Wnt8 in the presumptive mesoderm. The relevance of Wnt8 as a Snail target was tested by bypassing this repression in wild type embryos using a maternal Gal4 driver to drive UAS-Wnt8. This led to a loss of ventral furrow formation and a phenocopy of the snail mutant phenotype, thereby indicating that the repression of Wnt8 by Snail is important for gastrulation. Further investigation into the mechanism revealed a reduction in the expression of multiple target genes of Dorsal (including snail) in these Wnt8 overexpressing embryos. Ventral nuclear Dorsal protein was reduced as compared to wild type, suggesting that high levels of Wnt8 can antagonize Dorsal nuclear localization. Deficiency embryos lacking Wnt8 showed the opposite phenotype of expanded anterior and posterior snail RNA staining, as well as an expanded nuclear Dorsal signal in the posterior. This could be phenocopied using dsRNA against Wnt8, and was fully rescueable in the deficiency background using a Wnt8 genomic fragment. It has been reported that loss-of-function snail embryos lose the sharp lateral boundaries and high levels of snail expression more rapidly as compared to wild type. We hypothesize that this loss is due to derepressed Wnt8 antagonizing Dorsal and consequently its target, snail. In support of our hypothesis, double mutants of snail and the Wnt8 deficiency show a rescue of the snail pattern, though not a rescue of ventral furrow formation. Western blot analysis reveals a decrease in the levels of phosphorylation of Dorsal in Wnt8 overexpressing embryos as compared to wild type. Phosphorylation of Dorsal is required for its nuclear translocation. Hence, these data corroborate the observation of reduced nuclear Dorsal in embryos overexpressing Wnt8. Together, these data point to Wnt8 being an important target and a feedback inhibitor of the Dorsal/Twist/Snail pathway that achieves its effect by the inhibition of Dorsal.
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Humphrey, Peter Saah. "Signal transduction mechanisms for stem cell differentation into cardiomyocytes." Thesis, University of Hertfordshire, 2009. http://hdl.handle.net/2299/3760.
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Cardiovascular diseases are among the leading causes of death worldwide and particularly in the developed World. The search for new therapeutic approaches for improving the functions of the damaged heart is therefore a critical endeavour. Myocardial infarction, which can lead to heart failure, is associated with irreversible loss of functional cardiomyocytes. The loss of cardiomyocytes poses a major difficulty for treating the damaged heart since terminally differentiated cardiomyocytes have very limited regeneration potential. Currently, the only effective treatment for severe heart failure is heart transplantation but this option is limited by the acute shortage of donor hearts. The high incidence of heart diseases and the scarcity donor hearts underline the urgent need to find alternative therapeutic approaches for treating cardiovascular diseases. Pluripotent embryonic stem (ES) cells can differentiate into functional cardiomyocytes. Therefore the engraftment of ES cell-derived functional cardiomyocytes or cardiac progenitor cells into the damaged heart to regenerate healthy myocardial tissues may be used to treat damaged hearts. Stem cell-based therapy therefore holds a great potential as a very attractive alternative to heart transplant for treating heart failure and other cardiovascular diseases. A major obstacle to the realisation of stem cell-based therapy is the lack of donor cells and this in turn is due to the fact that, currently, the molecular mechanisms or the regulatory signal transduction mechanisms that are responsible for mediating ES cell differentiation into cardiomyocytes are not well understood. Overcoming this huge scientific challenge is absolutely necessary before the use of stem cell-derived cardiomyocytes to treat the damaged heart can become a reality. Therefore the aim of this thesis was to investigate the signal transduction pathways that are involved in the differentiation of stem cells into cardiomyocytes. The first objective was the establishment and use of cardiomyocyte differentiation models using H9c2 cells and P19 stem cells to accomplish the specific objectives of the thesis. The specific objectives of the thesis were, the investigation of the roles of (i) nitric oxide (ii) protein kinase C (PKC), (iii) p38 mitogen-activated protein kinase (p38 MAPK) (vi) phosphoinositide 3-kinase (PI3K) and (vi) nuclear factor-kappa B (NF-kB) signalling pathways in the differentiation of stem cells to cardiomyocytes and, more importantly, to identify where possible any points of convergence and potential cross-talk between pathways that may be critical for differentiation to occur. P19 cells were routinely cultured in alpha minimal essential medium (α-MEM) supplemented with 100 units/ml penicillin /100 μg/ml streptomycin and 10% foetal bovine serum (FBS). P19 cell differentiation was initiated by culturing the cells in microbiological plates in medium containing 0.8 % DMSO to form embryoid bodies (EB). This was followed by transfer of EBs to cell culture grade dishes after four days. H9c2 cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% FBS. Differentiation was initiated by incubating the cells in medium containing 1% FBS. In both models, when drugs were employed, they were added to cells for one hour prior to initiating differentiation. Cell monolayers were monitored daily over a period of 12 or 14 days. H9c2 cells were monitored for morphological changes and P19 cells were monitored for beating cardiomyocytes. Lysates were generated in parallel for western blot analysis of changes in cardiac myosin heavy chain (MHC), ventricular myosin chain light chain 1(MLC-1v) or troponin I (cTnI) using specific monoclonal antibodies. H9c2 cells cultured in 1% serum underwent differentiation as shown by the timedependent formation of myotubes, accompanied by a parallel increase in expression of both MHC and MLC-1v. These changes were however not apparent until 4 to 6 days after growth arrest and increased with time, reaching a peak at day 12 to 14. P19 stem cells cultured in DMSO containing medium differentiated as shown by the timedependent appearance of beating cardiomyocytes and this was accompanied by the expression of cTnI. The differentiation of both P19 stem cells and H9c2 into cardiomyocytes was blocked by the PI3K inhibitor LY294002, PKC inhibitor BIM-I and the p38 MAPK inhibitor SB2035800. However when LY294002, BIM-I or SB2035800 were added after the initiation of DMSO-induced P19 stem cell differentiation, each inhibitor failed to block the cell differentiation into beating cardiomyocytes. The NF-kB activation inhibitor, CAPE, blocked H9c2 cell differentiation into cardiomyocytes. Fast nitric oxide releasing donors (SIN-1 and NOC-5) markedly delayed the onset of differentiation of H9c2 cells into cardiomyocytes while slow nitric oxide releasing donors (SNAP and NOC-18) were less effective in delaying the onset of differentiation or long term differentiation of H9c2 cells into cardiomyocytes. Akt (protein kinase B) is the key downstream target of PI3K. Our cross-talk data also showed that PKC inhibition and p38 MAPK inhibition respectively enhanced and reduced the activation of Akt, as determined by the phosphorylation of Akt at serine residue 473. In conclusion, PKC, PI3K, p38 MAPK and NF-kB are relevant for the differentiation of stem cells into cardiomyocytes. Our data also show that the PKC, PI3K and p38 MAPK signalling pathways are activated as very early events during the differentiation of stem cells into cardiomyocytes. Our data also suggest that PKC may negatively regulate Akt activation while p38 MAPK inhibition inhibits Akt activation. Our fast NO releasing donor data suggest that nitric oxide may negatively regulate H9c2 cell differentiation.
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Hsioa, Tingfen, and 蕭婷分. "Identification and characterization of potential Wnt signaling inhibitors." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/3d2jkh.
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碩士輔仁大學
生命科學系碩士班
100
Hepatocellular carcinoma (HCC) is one of the leading cancers in the world with the mortality of more than half a million deaths per year. Using the platform of Connectivity Map (cmap), which hosts a much greater number of gene expression profiles from culture human cancer cell lines treated with bioactive small molecular, we have identified some drugs may reverse the gene expression signature of HCC and have potentials for treating HCC. Due to the inappropriate activation of Wnt/-catenin signaling pathway in HCC, this thesis prompt to identify the drugs that selected from cmap can also inhibit Wnt/-catenin signaling pathway. Using HCC cells containing β-catenin/Tcf-response reporter, we first screen the drug as potential inhibitor of Wnt/β-catenin pathway. Here, four drugs named 8-azaguanine, thiostrepton, withaferin A and antimycin A have been identified as Wnt/-catenin signaling pathway inhibitors. In addition, dosage-dependent inhibition of Wnt/-catenin signaling pathway can also be demonstrated in two different HCC cells. Moreover, these four drugs show different degree of inhibition on the proliferation of Huh7 and Mahluvu cells. To investigate these drugs mechanistically, we have examine the effects of these potential Wnt/-catenin signaling inhibitors on the expression of -catenin, c-myc and cyclin D1. We find that although 8-azaguanine and thiostrepton can not reduce the protein level of -catenin, they reduce the expression of c-myc and cyclin D1. Since cytosolic -catenin slightly increase, while nucleic -catenin decrease under drug treatment, we conclude that 8-azaguanine and thiostrepton may prohibit the nuclear entry of -catenin, which conduct the inhibition of Wnt/-catenin signaling. Alternatively, withaferin A and antimycin A treatment can decrease the protein level of -catenin, indicating they may promote the degradation of -catenin and abolish following signaling. In addition, withaferin A and antimycin A treatment can increase the protein level of LC3-II, which is a marker of autophagy, and decrease pro-caspase-3. Further studies should be performed to ask whether autophagy play crucial roles in withaferin A and antimycin A conducted cell death. Overall, this thesis has identified four drugs as Wnt/-catenin signaling inhibitors and may serve as potential drugs for treating HCC.
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Yang, Chih-Yu, and 楊智宇. "Wnt/β-catenin Signaling Inhibitors in the Pathogenesis of Uremic Ectopic Ossifications." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/6pq969.
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博士國立陽明大學
臨床醫學研究所
103
Background: Uremic patients manifest an accelerated phenotype of soft bone and hard artery. The process of “hard artery”, i.e. vascular calcification, has been associated with the canonical Wnt/β-catenin signaling pathway in cell cultures and animal studies. On the other hand, the uremic “soft bone”, i.e. osteomalacia, has been seen in the alveolar bone, but radiographic evidence has shown that the adjacent pulp chamber is reciprocally obliterated; however, this has not been supported by any pathological evidence. We speculated that Wnt/β-catenin signaling pathway might participate in the pathogenesis of uremic ectopic ossifications of soft tissues including both dental pulp and vasculature. Methods: We used a uremic rat model with secondary hyperparathyroidism induced by 5/6 nephrectomy surgery and high-phosphate diet to examine the dental pulp and adjacent alveolar bone pathology. In addition, we collected pulp tissues for real-time polymerase chain reaction. Meanwhile, because the relationship between Wnt/β-catenin signaling inhibitors and vascular calcification is unknown in uremic patients, we investigated the associations between serum dickkopf-1 (Dkk-1) and sclerostin, two circulating inhibitors of the Wnt/β-catenin signaling pathway, and the severity of aortic calcification (AoC) and cardiovascular outcomes in uremic patients. Results: We found an opposite histopathological presentation of the ossified dental pulp and the osteomalacic adjacent alveolar bone. Furthermore, pulp cells with positive staining for Thy-1, a surrogate stem cell marker, were significantly reduced in the pulp of uremic rats compared to the controls, indicating a paucity of stem cells. This was further evidenced by the reduced pulp expression of dickkopf-1 (Dkk-1), a Wnt/β-catenin signaling inhibitor produced by mesenchymal stem cells. In contrast, expressions of receptor activator of nuclear factor κB ligand (RANKL) and RANK in uremic pulp were up-regulated, probably to counteract the ossifying process of uremic pulp. As for the uremic patients, the circulating sclerostin level was inversely associated with the severity of AoC (p = 0.035) and indicators of the bone turnover rate including serum alkaline phosphatase (ALP) (r = -0.235, p = 0.008) and intact parathyroid hormone (r = -0.523, p < 0.001). Furthermore, Cox regression analysis indicated that the patients with high circulating sclerostin levels were less likely to experience future cardiovascular events [1 pmol/L sclerostin increase, hazard ratio 0.982 (95% CI, 0.967-0.996), p = 0.015] after adjusting for a propensity score. Conclusions: Uremic pulp ossifications were associated with a paucity of stem cells and dysregulated Dkk-1 and RANKL signaling systems, further shifting the imbalance toward osteogenesis. Additionally, in uremic patients, circulating sclerostin is inversely associated with AoCs and future cardiovascular events. Our findings suggest that Wnt/β-catenin signaling inhibitor deficit is associated with both pulp ossification and vascular calcification in uremic subjects. Meanwhile, sclerostin, as a bone-related protein, might act as a communicator between uremic bone and vasculature. Strategies to modulate Wnt/β-catenin signaling inhibitors may offer a therapeutic potential to improve dental and vascular health in uremic patients.
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Tschirschmann, Miriam [Verfasser]. "Regulation des WNT-Inhibitors DKK1 im In-vitro-Kondensationsmodell / vorgelegt von Miriam Tschirschmann." 2009. http://d-nb.info/999611216/34.
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Perusini, Stephen John. "High-thoughput Screen to Identify Small Molecule Inhibitors of the Canonical Wnt Signaling Pathway." Thesis, 2008. http://hdl.handle.net/1807/17210.
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Wnt signaling is important in human development and disease, thus dysregulated beta-catenin constitutes an attractive target for drug intervention. The few functional inhibitors currently available target transcriptional activation, therefore, identifying novel upstream modulators would be of tremendous importance to elucidating the mechanisms involved in regulatingbeta-catenin activity.
To achieve this, I developed a high-throughput screen to assess beta-catenin stability in mammalian cells using a luciferase tagged beta-catenin molecule. This assay was used to screen three chemical libraries to identify small molecule modulators of the pathway. Identified inhibitors/activators of the pathway were investigated via secondary assays. The most promising inhibitor, 21H7, significantly attenuated activated beta-catenin signaling in colon cancer cells, decreasing beta-catenin stability. The inhibitory effects of 21H7 and a structurally similar compound were shown to not only inhibit Wnt target gene expression in colon cancer cells, but also prostate cancer lines. Thus, 21H7 represents an attractive lead compound for further study.
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Janečková, Lucie. "Signální dráha Wnt v obnově a tumorigenezi střevního epitelu." Doctoral thesis, 2014. http://www.nusl.cz/ntk/nusl-338480.
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The canonical Wnt signaling pathway is one of the most important pathways involved in cell proliferation and differentiation. It is highly conserved in evolution and participates not only in embryonic development but also in adult tissue homeostasis. In the intestine, Wnt signaling is closely connected to maintenance of intestinal stem cells and renewal of the epithelia. Conversely, aberrant activation of the Wnt signaling pathway underlies different types of human diseases. Its constitutive activation results in neoplasia and specifically in development of colorectal cancer, which is the third most common malignancy in western world. The aim of this thesis was to uncover various aspects of the regulatory mechanisms of the Wnt/β-catenin signaling cascade. Furthermore, I headed to find novel Wnt pathway modulators and confirm their function in vivo. The results are presented in four publications. The first study examines murine Wnt proteins processing and the sequential order of Wnt post-translational modifications which are required for the secretion and signaling activity of the ligands. Next publication focuses on the gene Troy, which we identified as negative regulator of Wnt signaling. TROY was discovered as a Wnt target gene during DNA microarray profiling of human colorectal cancer cells....
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Fauser, Jane Kathryn. "Medium chain fatty acids and Wnt/β-Catenin inhibitors as adjunctive colorectal cancer chemotherapeutic agents." Thesis, 2013. http://hdl.handle.net/2440/94400.
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Chemotherapy remains a primary treatment for advanced stage colorectal cancer (CRC). Although more targeted chemotherapeutic agents are under development, currently prescribed cytotoxic agents target rapidly-dividing cells without discrimination between neoplastic and non-neoplastic cells, inducing debilitating side-effects with high morbidity and mortality rates. The development of less injurious chemotherapeutic agents holds promise in alleviating the negative side-effects of chemotherapy. Fatty acids (FAs) are bioactive aliphatic monocarboxylic acids categorized by the number of carbon atoms in the aliphatic chain, classified as short chain fatty acids (SCFAs) (< C8:0), medium chain fatty acids (MCFAs) (C8-14:0) and long chain fatty acids (LCFAs) (>C16:ɷ3-9). Exogenous applications of FAs has demonstrated anti-neoplastic properties, and FA have been suggested as adjunctive chemotherapeutic agents for the treatment of CRC. SCFAs and LCFAs have demonstrated potent anti-neoplastic properties in in vitro models of CRC. However, there are gaps in the literature regarding the potential anti-neoplastic properties of MCFAs in CRC. Endogenous adjunctive CRC therapies manipulate signalling pathways related to the instigation and progression of CRC, and have only recently been incorporated into standard chemotherapeutic protocols. The Wnt/β-catenin signalling pathway is dysregulated in the majority of CRC cases leading to up-regulation of cell proliferation, crypt expansion and mutated intestinal stem cells (ISC). Inhibition of this pathway has demonstrated a down-regulation of cell proliferation. However, the effect on the process of crypt fission and ISC expression is unknown. In vitro (cell culture) technologies are underutilised in investigations of the biological effect and mechanisms of actions of pharmacological agents, bioactive agents and anti-neoplastic agents, when related to intestinal function. There is a paucity of intestinal non-transformed cell lines due to difficulties in the derivation and culture of these
cells. This thesis investigates the effects of a MCFA on a CRC cell line (Caco-2) and a non-neoplastic intestinal cell line (IEC-6), and the in vivo endogenous application of Dickkopf-1 (Dkk-1), a Wnt signalling pathway inhibitor. Furthermore, this thesis describes the derivation of a short term porcine small intestinal cell line.
Initially in Chapter 2, Caco-2 cells were treated with increasing concentrations of the MCFA, Lauric acid (LA) (C12:0), with cell death compared to the established anti-neoplastic SCFA, butyrate (C4:0) to induce cell death or cytotoxicity, using a CRC in vitro model. This study demonstrated the novel finding that LA exerts cytotoxic properties at doses of 0.5mM and 1mM, having induced a significant reduction in cell viability in Caco-2 cells, compared to butyrate and negative controls, as ascertained by trypan blue exclusion assay. This data is supported by published cytotoxic studies in lymphoblastic cells. It has been proposed that anti-neoplastic properties of FAs are carbon atom chain length dependent. On this basis, preferential cytotoxic properties of MCFAs of differing carbon atom chain lengths were investigated. LA exerted preferential cytotoxic properties compared to C10:0 and C14:0 MCFAs. Few studies simultaneously investigate the potential negative side-effects of FAs on non-neoplastic intestinal cell lines. In Chapter 2, the murine small intestinal cell line, IEC-6 was treated with cytotoxic doses of LA. LA induced significant cell death compared to butyrate and negative control cells. This data supported further investigation into the mechanisms underpinning cell death induced by LA in Caco-2 and IEC-6 cell lines.
Using flow cytometric analysis compared to butyrate, Chapter 3 demonstrated that cytotoxic doses of LA induced a significant increase in the percentage of cells undergoing apoptosis as opposed to necrosis in Caco-2 cells. The new discovery that LA induced apoptosis in Caco-2 cells supported further exploration into the anti-neoplastic mechanisms of LA. Few studies have explored the effects of intracellular redox modulation of intestinal cell homeostasis, thus this study investigated the effect of LA on reduced glutathione (GSH) levels as measured by enzymatic analysis, ROS generation and modification to cell cycle phases were measured using flow cytometric analysis. It was determined that LA reduced the availability of GSH, thereby demonstrating that LA modulated the redox system of the Caco-2 cell line. A significant increase in levels of ROS was detected in LA-treated Caco-2 cells compared to butyrate, indicating that LA induced a higher oxidative state than butyrate. Therefore, the reduction in all phases of the cell cycle in LA treated Caco-2 cells may not have been the key contributor to the induction of apoptosis. Butyrate induced significant apoptosis in Caco-2 cells compared to PBS-treated controls, reduced GSH levels equivalent to LA, decreased cells in G0/G1 phases and generated less ROS than LA. These different effects are proposed to be due to the different carbon atom chain lengths of butyrate and LA. LA at cytotoxic doses significantly reduced cell viability in IEC-6 cells, associated with a reduction in GSH, generation of ROS and modification to all cell cycle phases. Butyrate on the other hand did not decrease IEC-6 viability, reduce GSH, modify phases of the cell cycle or generate ROS, indicating that this SCFA did not influence the redox system of this cell line. This may have been related to the genetic differences in non-transformed (p53 and Wnt positive wild type) and transformed cell lines (p53 and Wnt signalling mutant).
Mutations in Wnt/β-catenin are present in the majority of CRC cases, resulting in over-expression of the downstream molecule, β-catenin, inducing uncontrolled cell proliferation leading to up-regulation of crypt fission, a precursor and promoter of CRC polyps, thereby generating a greater number of crypts which produce mutated intestinal stem cells to populate CRC tumours. Dickkopf (Dkk-1) is a pan negative regulator of the Wnt/β-catenin signalling system. In Chapter 4 neonatal rats (day 11-15) were treated with increasing doses of Dkk-1 (30ng and 100ng). A microdissection technique in the small intestine demonstrated that both doses of Dkk-1 reduced villus and crypt areas significantly. Therefore, it was concluded that Dkk-1 reduced crypt fission but not crypt hyperplasia in neonatal rats. The Wnt controlled intestinal stem cell (ISC) marker Lgr5 mRNA was significantly reduced in Dkk-1 treated animals in both the small and large intestine. Expression of other ISC markers, DCAMKL-1, Bmi1 and OLFM4 mRNA remained unchanged in the small intestine. In the large intestine the ISC marker DCAMKL-1 mRNA expression remained unchanged. Bmi1 and OLFM4 mRNA expression was significantly reduced. Protein expression of the Wnt downstream effector molecule, β-catenin, as measured by immunohistochemistry, was significantly reduced in the small intestine of Dkk-1 treated animals.
Rapid and easily reproducible cell culture models are essential for expanding studies of intestinal function and investigations into pharmacological and nutraceutical agents, concomitantly reducing the use of research animals. In Chapter 5 a hybrid of intestinal primary cell isolation methods was used to derive a short-term porcine small intestinal cell line. Cells were isolated from the jejunum and ileum of a stillborn large white piglet, and, for the first time, maintained in culture for a 5 week period before reaching senescence. This demonstrates that a non-transformed epithelial cell line can be generated using basic cell culture practices, leading to the increased capacity to evaluated bioactive molecules on normal intestinal epithelial cells.
In conclusion, this thesis has provided new evidence into the anti-neoplastic properties of MCFAs in a CRC model. The MCFA, LA modulated the intracellular redox system, inducing apoptosis in Caco-2 cells, with short carbon atom chain FAs inducing a lesser degree of cell death. This finding is important as it demonstrates that carbon atom chain length influences the rate of apoptosis and possibly the mechanisms underpinning this cytotoxicity. Thus, FAs hold promise to augment adjunctive chemotherapeutic agents. It is essential that FAs of all carbon atom chain lengths are evaluated both in vitro and in vivo for anti-neoplastic potential. Another key finding of this thesis was that crypt fission is negatively regulated by inhibition of the Wnt signalling system, critical in reducing the number of neoplastic adenomas and mutated ISC populations. This finding has potential for the use of negative regulators of the Wnt/β-catenin signalling system as possible adjunctive CRC chemotherapeutic agents. Finally, a new method to derive an intestinal cell line was developed. This simple method opens up wider scientific opportunities to evaluate the effects of bioactive compounds on species-specific non-transformed intestinal cells.Thesis(Ph.D.)-- University of Adelaide, School of Animal and Veterinary Sciences, 2013
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Pospíchalová, Vendula. "Nádorový supresor HIC1- nový inhibitor signalní dráhy Wnt." Master's thesis, 2008. http://www.nusl.cz/ntk/nusl-292043.
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1. ABSTRACT In all metazoan organisms Wnt ligands regulate various developmental and physiological processes during embryogenesis and also in adult tissues. Moreover, deregulation in Wnt-related signalling is associated with several human disorders including cancer. In the so-called canonical pathway, activation of the Wnt receptor complex Frizzled/LRP triggers a complex network of molecular events that ultimately lead to the stabilization of β-catenin and formation of TCF/β- catenin heterodimers in the nucleus. These protein complexes drive expression of a specific set of genes that control the cell fate decision. The Wnt pathway is tightly regulated by several mostly negative feedback loops involving scores of extracellular, cytoplasmic or nuclear proteins. Recently, we have identified tumour suppressor Hypermethylated in cancer 1 (HIC1) as a negative modulator of canonical Wnt signalling. The HIC1 gene encodes a BTB/POZ-zinc finger transcriptional repressor. Interestingly, the HIC1-dependent repression of the TCF/β-catenin- dependent genes is not mediated by direct association of HIC1 with the regulatory regions of the Wnt-responsive genes but rather by a sequestration of TCF/β-catenin complexes to the nuclear speckle-like structures - the HIC bodies. These data indicate quite a pleiotropic role of HIC1...
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Curtis, Courtney Lee. "Wnt signaling in zebrafish fin regeneration : chemical biology using a GSK3β inhibitor." Thesis, 2014. http://hdl.handle.net/1805/4835.
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Indiana University-Purdue University Indianapolis (IUPUI)Bone growth can be impaired due to disease, such as osteoporosis. Currently, intermittent parathyroid hormone (PTH) treatment is the only approved therapy in the United States for anabolic bone growth in osteoporosis patients. The anabolic effects of PTH treatment are due, at least in part, to modulation of the Wnt/β-catenin pathway. Activation of the Wnt/ β-catenin pathway using a small molecule inhibitor of GSK3β was previously shown to increase markers of bone formation in vitro. Our study utilized a zebrafish model system to study Wnt activated fin regeneration and bone growth. Wnt signaling is the first genetically identified step in fin regeneration, and bony rays are the main structure in zebrafish fins. Thus, zebrafish fin regeneration may be a useful model to study Wnt signaling mediated bone growth. Fin regeneration experiments were conducted using various concentrations of a GSK3β inhibitor compound, LSN 2105786, for different treatment periods and regenerative outgrowth was measured at 4 and 7 days post amputation. Experiments revealed continuous low concentration (4-5 nM) treatment to be most effective at increasing regeneration. Higher concentrations inhibited fin growth, perhaps by excessive stimulation of differentiation programs. In situ hybridization experiments were performed to examine effects of GSK3β inhibitor on Wnt responsive gene expression. Experiments showed temporal and spatial changes on individual gene markers following GSK3β inhibitor treatment. Additionally, confocal microscopy and immunofluorescence labeling data indicated that the Wnt signaling intracellular signal transducer, β-catenin, accumulates throughout GSK3β inhibitor treated tissues. Finally, experiments revealed increased cell proliferation in fin regenerates following LSN 2105786 treatment. Together, these data indicate that bone growth in zebrafish fin regeneration is improved by activating Wnt signaling. Zebrafish Wnt signaling experiments provide a good model to study bone growth and bone repair mechanisms, and may provide an efficient drug discovery platform.
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Chen, Yung-Fu, and 陳永富. "Modeling of Kinetics of the Wnt-EGFR Signaling Pathway and Inhibitor Effects on its Kinetic Behaviors." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/91322219733868287084.
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碩士國立臺灣師範大學
化學系
100
The Wnt and EGFR signaling pathways are known to relate to cell proliferation, differentiation, and apoptosis. Deregulation of these signaling pathways were found in various kinds of cancers. Toward better understanding of these two pathways, we used computer modeling method to model kinetics of these pathways. Based on currently available models, we expanded the model in order to include more effects in kinetics of these pathways and correlate with available experimental data. First, we added a negative feedback loop on the EGFR pathway which has inhibition effect on Raf-1 by ERKpp. When the K1 value of Raf-1 inhibition reaction was set in the range of 0.01 to 10, the computations gave that addition of this loop results in two different effects in the two models we used. The negative feedback loop has a little effect on ERKpp level in the model which includes Braf. In contrast, the negative feedback loop makes the level of phosphorylated ERK go down in the model without Braf. Second, a crosstalk between EGFR and Wnt pathways was added and kinetic modeling gave that：in the case this is a positive feedback loop, it induces the switch-like behavior of ERKpp expression by varying the concentration of the added factor Y when the β -catenin was not overexpressed. In the case it is a negative feedback loop, due to the stronger v negative feedback effect, that during Wnt signal stimulate the concentration of ERKpp has an oscillation behavior with larger amplitude during the period of Wnt signal stimulation. After that, concentration of ERKpp falls back to low level quickly. In addition, we investigated effect of adding kinase inhibitor(s) on the level of phosphorylated ERK (ERKpp) under the condition of β-catenin overexpression plus undergoing a wnt signal transient stimulation. In the case of adding one kinase inhibitor, the modeling gave that：kinase inhibitor of multiple-targets of the same strength have less inhibitory effect than inhibitor of singlet-target of Raf-1 kinase. Reduction of concentration of ERKpp was as small as to 1/6 fold only compared with the case of singlet-target in the examined model. Furthermore, we investigate effects of multiple-target inhibitors inhibiting Raf-1 and MEK kinases. It was found that in the case the ratio of Raf-1 inhibitor concentration to MEK inhibitor concentration is larger than 1.5, the inhibitor effect is better than one inhibitor of multiple-target with the same inhibition strengths. These results deepen our understanding of these two pathways and should be useful for future multiple-target kinase inhibitor design.
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Hsiung, Shih-Chieh, and 熊式潔. "Clozapine Inhibits Colorectal Cancer Cell Growth via Wnt/β-catenin and p21-Dependent Pathways." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/q5p3ra.
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碩士國立臺灣海洋大學
生物科技研究所
101
Clinical studies have shown that patients with schizophrenia have a lower cancer incidence than the general population. Several antipsychotics have been demonstrated to confer cytotoxic effects on cancer cells, but the detailed molecular mechanism is unclear. Abnormal activation of the Wnt/β-catenin pathway plays an important role in colorectal cancer. In this study, two human colorectal cancer cell lines, HCT116 and SW480, were used to investigate the effect of clozapine, an antipsychotic widely used to treat patient with refractory schizophrenia, on Wnt/β-catenin pathway. Preliminary data showed that clozapine inhibited proliferation of SW480 and HCT116 cells in time- and dosage-dependent manners. Clozapine suppressed -catenin mRNA and protein levels. Furthermore, clozapine also decreased the expression of β-catenin downstream target gene cyclin D1. These results suggested that clozapine inhibited the growth of colorectal cancer cells through the inhibition of β-catenin transcription. On the other topic, our previous results revealed that clozapine increased p21 protein expression and reactive oxygen species (ROS) in HCT116 cancer cells. Studies have shown that p21 protects cells against oxidative stress. Here, we examined the relationship between clozapine-induced ROS and p21. Clozapine inhibited cell proliferation and colony formation via p53-independent manner and HCT116 p21-/- cells were more sensitive to clozapine. Clozapine increased more ROS and induced apoptosis in HCT116 p21-/- cells compared to HCT116 cells. In HCT116 cells, a ROS scavenger vitamin E (α-Tocopherol) decreased ROS, but also reduced p21 protein expression. The results showed that clozapine-increased ROS caused p21 protein expression. Furthermore, we also demonstrated that p21-mediated ROS is associated with Nrf2, a transcription factor can activate an antioxidant response to decrease ROS. It needs to further verify whether clozapine induces apoptosis via p21 and Nrf2 to control ROS levels.
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Miller, Catherine A. "Shifted, the Drosphila homolog of the Wnt inhibitory factor 1, is involved in the Hedgehog signaling pathway." 2004. http://www.library.wisc.edu/databases/connect/dissertations.html.
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HUANG, SHIH-YU, and 黃詩于. "Resveratrol Enhance Paclitaxel and Doxorubincin to Inhibits Human Adenocarcinoma (A549) Cell Proliferation through Wnt pathway." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/qwj3es.
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Becker, Marco. "Wif1 Inhibits the Growth of Basal Cell Carcinoma." Doctoral thesis, 2015. http://hdl.handle.net/11858/00-1735-0000-0028-867F-F.
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