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Title: Studies on furin as target for possible therapeutic intervention in tumor development
Other Titles: Studies over furine als doelwit voor mogelijke therapeutische interventie in de ontwikkeling van tumoren
Authors: Zhu, Jingjing; S0211445
Issue Date: 10-Sep-2013
Abstract: Summary Proprotein convertases (PCs) form a group of serine endoproteases that are essential for the endoproteolytic processing of proproteins into their active forms. Some PCs have been proposed to be potential therapeutic targets for cancer intervention because elevated PC activity has been observed in many different cancer types and because many of the PC substrates, such as pro-IGF-1R, pro-TGF-ß, pro-VEGF, are involved in signaling pathways related to tumor development. In this study, we have developed furin-specific nanobodies for the inhibition of furin activity. Antibody-based inhibitors would provide a highly specific strategy for targeted inhibition. Compared to conventional antibodies, nanobodies are 10 times smaller in size but possess the same antigen binding affinity. They are also easy to produce in bulk quantities at low costs and to engineer. We have developed and characterised 13 different nanobodies. Four of them, when expressed in mammalian cells, show furin inhibiting capacity. This inhibitory effect is strictly specific for furin. After expression of these four nanobodies in E coli and subsequent purification, their enzyme inhibitory capacities were also evaluated in vitro. It has been demonstrated that they probably inhibit enzyme activity by inducing a conformational change of the enzyme, thereby causing steric hindrance for substrates to enter the catalytic site. Inhibitory activity was found only when large protein substrates were used. No inhibition could be observed when a small fluorogenic peptide substrate was tested. The purified nanobodies showed cell protective effect against diphtheria toxin and anthrax toxin. These effects are valuable and should be investigated further in vivo since it indicates the nanobodies could become valuable anti-bacterial tools. Similarly, they might also constitute valuable anti-viral reagents, since they are likely to inhibit processing of viral envelope glycoproteins critical for infectivity. Purified nanobodies do not lead to the inhibition of furin in the trans-golgi network. A possible explanation for this phenomenon probably lies in their insufficient penetration into cells. On one hand, further studies could focus on improving cell penetration capabilities of the nanobodies, on the other hand, a gene therapy approach might be contemplated using the nanobody cDNA since overexpression of nanobodies in mammalian cells led to promising inhibitory effects regarding the maturation of substrates which have been shown to be important in cancer development, for example pro-TGF-ß and pro-GPC3.In this study, we also evaluated small molecule polyphenols as potential furin inhibitors. We have chosen polyphenols for several reasons. Firstly, they are food components or food additives. Therefore, they are not likely to cause deleterious effects in the general population. Secondly, many of them have been claimed to exert anti-cancer effects. Several polyphenols have been evaluated by other research groups. Majumdar and co-workers (Majumdar et al, 2010), for instance, reported about in vitro inhibitory activity towards furin and the authors consequently suggested that the anti-cancer effects of these polyphenols might be related to their furin inhibitory activity. Our attempts to measure the furin inhibitory effect of a series of polyphenols resulted in confirmation of the in vitro furin inhibition. However, these inhibitory effects could not be substantiated in living cells undermining the furin inhibition basis for the anti-cancer claim. The in vitro fluorimetric assay, therefore, leads to results that not always have a predictive value for the in vivo situation. The in vitro test thus easily leads to false positive results. In the present study, the false positive effects of the main component of green tea (-)EGCG on the PC furin were studied in greater detail. It was first demonstrated that the false positive effects were not related to aggregation of the polyphenols. Detergent-based enzyme assays are in use to limit false positive effects related to enzyme unfolding caused by adsorption of the enzyme to aggregating small molecules. Furthermore, polyphenols in solution, such as catechins, may form reactive compounds when exposed to air and these can bind to enzymes with enzyme inhibition as a result. This should be taken into account in screening assays. Adding reduced glutathione to the detergent-based assay, as used in these studies, to measure furin processing activity strongly reduced inhibition by a number of polyphenols (catechins, gallic acid, and quercetin), while the effect on the genuine inhibitor nona-D-arginine remained unchanged. In conclusion, the combined use of detergent and glutathione in an in vitro screening assay for furin inhibitors improves the predictive value. Besides improving the in vitro assay, in this study, we have further investigated the possible indirect inhibitory effect of polyphenols against endogenous furin activity in a cellular environment. Six out of twenty-seven different polyphenols appear to inhibit furin indirectly in such an assay. The inhibitory effects of four compounds were further evaluated. All four compounds appeared to inhibit zymogen maturation and consequently PC activity. The molecular mechanism for the inhibitory activity of one of them, curcumin, was elucidated at the subcellular level in experiments with mammalian cells. We demonstrated that curcumin’s inhibitory effect on Ca2+ uptake into the ER by the enzyme sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) allows and is sufficient to explain the inhibitory effect on PC activity. The inhibition of PC activity by all four compounds resulted in a reduction of anchorage-independent growth (a hallmark of malignancy) of cancer cells in culture. Targeted drug delivery approaches could reduce potential deleterious side effects in therapeutic use. In this study, we have therefore evaluated a nanoparticle drug carrier for its potential for targeted drug delivery. Magnetic nanoparticles (MPs) loaded with an anti-tumor drug, in combination with external magnetic field (EMF)-guided delivery, can improve the efficacy of treatment and may decrease side effects. Our results show that our simple and adaptive nanoparticle design may accommodate chemotherapy for drug delivery optimization. This could also increase the margin of safety in future treatment of solid tumors with furin inhibitors.
Table of Contents: ACKNOWLEDGEMENT I
INDEX V
LIST OF ABBREVIATIONS VII
1. GENERAL INTRODUCTION 1
1.1. Furin and its family members 3
1.1.1. Overview of the PC family 3
1.1.2. PC structure 4
1.1.3. The catalytic mechanism of PCs 6
1.1.4. Proprotein convertase cleavage sites 7
1.2. Localization and expression of furin and other PCs 7
1.3. PC maturation 8
1.4. Furin and other PCs are important in pathophysiology 9
1.4.1. The role of furin and other PCs in cancer 9
1.4.2. The role of furin and other PCs in other diseases 14
1.5. PC inhibitors 17
1.5.1. Protein-based inhibitors 17
1.5.2. Peptide-derived inhibitors 18
1.5.3. Small molecule-based inhibitors 18
1.5.4. Antibody-based inhibitors 19
1.6. Conclusions 20
2. RESEARCH OBJECTIVES 23
3. RESULTS 25
3.1 Generation and characterization of non-competitive furin-inhibiting nanobodies 25
3.2 Limitations of inhibitory activities of polyphenols on furin-mediated substrate processing 39
3.3 Polyphenols can inhibit furin in vitro as a result of the reactivity of their auto-oxidation products to proteins 51 错误!未定义书签。
3.4 Polyphenols with indirect proprotein convertase inhibitory activity 65
3.5 Curcumin inhibits proprotein convertase zymogen maturation 77
3.6 A novel magnetic nanoparticle drug carrier for enhanced cancer chemotherapy 95
4. GENERAL DISCUSSION 105
SUMMARY 113
SAMENVATTING 117
BIBLIOGRAPHY 121
CURRICULUM VITAE 131
ISBN: 9789090277561
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Laboratory of Molecular Oncology (-)
Department of Human Genetics - miscellaneous

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