doktorska disertacija
Ana Mitrović (Author), Janko Kos (Mentor)

Abstract

Poveĉano izraţanje in aktivnost lizosomskih cisteinskih karboksipeptidaz, katepsinov B in X, je v telesu povezano s številnimi patološkimi procesi, vkljuĉno z nastankom in napredovanjem raka. Pri slednjem sta katepsina B in X udeleţena pri razgradnji zunajceliĉnega matriksa (ZCM), migraciji, invaziji, metastaziranju in angiogenezi. Pri napredovanju tumorjev ima pomembno vlogo tudi epitelno-mezenhimski prehod (EMT), kjer pa udeleţenost katepsinov B in X ni natanĉno poznana. V okviru doktorske disertacije smo zato v prvem delu raziskali udeleţenost katepsinov B in X pri EMT in razloţili mehanizme njunega delovanja pri tem procesu. Z uporabo tumorskih celiĉnih linij, ki se razlikujejo v izraţanju epitelijskih in mezenhimskih oznaĉevalcev in morfologiji celic, smo pokazali, da so višje koncentracije katepsinov B in X povezane z invazivnim mezenhimskim celiĉnim fenotipom in spodbujajo EMT. Nasprotno lahko obraten proces mezenhimsko-epitelnega prehoda sproţimo z njunim hkratnim utišanjem. Kot moĉnejši promotor EMT se je izmed obeh katepsinov izkazal katepsin B. Na epitelijski celiĉni liniji raka dojke MCF7 smo spremljali tudi spremembe v izraţanju katepsinov B in X po sproţitvi EMT s transformirajoĉim rastnim dejavnikom β1 (TGF-β1), ĉigar signalna pot je ena kljuĉnih signalnih poti, ki sproţi EMT pri raku. TGF-β1 je poveĉal izraţanje in aktivnost katepsina B, izraţanje katepsina X pa ni odvisno od TGF-β1. Dobljeni rezultati tako opredeljujejo EMT kot dodatni mehanizem, preko katerega katepsina B in X prispevata k napredovanju tumorjev. Z zmanjšanem njunega izraţanja ali inhibicijo encimske aktivnosti lahko tako tumorskim celicam povrnemo manj agresiven epitelijski celiĉni fenotip. Katepsina B in X sta zaradi svoje pomembne vloge pri številnih procesih pri raku primerni tarĉi za razvoj novih protitumornih uĉinkovin. V naslednjem sklopu doktorata smo se zato osredotoĉili na identifikacijo novih nizko molekularnih spojin, ki bi selektivno in reverzibilno zavirale aktivnost katepsinov B in X ter bi bile primerni kandidati za nadaljnja testiranja. Katepsin B ima, zaradi prisotnosti dodatnega strukturnega elementa, imenovanega zapora zanka, poleg karboksipeptidazne (eksopeptidazne) aktivnosti, tudi endopeptidazno aktivnost. Ravno za slednjo je veljalo, da je odgovorna za škodljivo delovanje katepsina B pri raku. V nadaljevanju doktorske disertacije smo z uporabo novih selektivnih sinteznih inhibitorjev katepsina B 2-{[(8-hidroksi-5-nitrokinolin-7-il)metil]amino}-acetonitril (1), 8- (4-metilpiperidin-1-il)-5-nitrokinolin (2) in 7-[(4-metilpiperidin-1-il)metil]-5-nitrokinolin-8-ol (3) ovrednotili vpliv obeh aktivnosti katepsina B na razgradnjo ZCM in tumorsko invazijo. Pri tem smo pokazali, da obe aktivnosti katepsina B – tako endopeptidazna kot eksopeptidazna – prispevata k razgradnji ZCM in invaziji tumorskih celic. Inhibitorji, ki bi hkrati moĉno inhibirali obe aktivnosti, bi bili uporabni v protitumorski terapiji. V in vitro in in vivo tumorskih modelih smo ovrednotili protitumorno delovanje nitrokoslina, znane protimikrobne uĉinkovine, ki je bil v predhodni študiji identificiran kot uĉinkovit selektivni inhibitor endopeptidazne aktivnosti katepsina B. Na izbranih tumorskih in endotelijskih celiĉnih linijah, ki izraţajo veĉje koliĉine katepsina B, je nitroksolin znaĉilno zmanjšal zunajceliĉno razgradnjo DQ-kolagena IV, invazijo tumorskih celic, kot tudi rast in invazijo veĉceliĉnih tumorskih sferoidov ter tvorbo endotelijskih cevk v modelu angiogeneze. Prav tako je znaĉilno zmanjšal rast tumorjev, angiogenezo in metastaziranje in vivo v treh neodvisnih mišjih tumorskih modelih. Izsledki raziskave tako opredeljujejo nitroksolin kot obetavno novo zdravilo za zdravljenje raka. V nadaljevanju smo ovrednotili še protitumorno delovanje derivata nitroksolina z 2-(etilamino)acetonitrilnim substituentom na mestu 7 (spojina 17), ki je v predhodni študiji v testih encimske kinetike izkazoval znaĉilno boljšo inhibicijo endopeptidazne aktivnosti katepsina B v primerjavi z nitroksolinom. Spojina 17 je bolj uĉinkovito zmanjšala tudi invazijo tumorskih celic in njihovo migracijo. Hkrati je v primerjavi z nitroksolinom bolj uĉinkovito zmanjšala tudi rast tumorjev in vivo na LPB mišjem modelu fibrosarkoma. Z dodatkom 2-(etilamino)acetonitrilne skupine na molekulo nitroksolina smo tako uspeli izboljšati njegove farmakološke lastnosti in pridobiti novo, še bolj uĉinkovito spojino, ki je kandidat za protitumorno terapijo. V naših raziskavah smo kot uĉinkovit inhibitor katepsina B med drugim identificirali tudi kompleks kliokinola z rutenijem [Ru(η6-p-cimen)(Cq)Cl]. Kompleks kliokinola z rutenijem v nizkih koncentracijah ni vplival na viabilnost celic, je pa znaĉilno zmanjšal razgradnjo proteinov ZCM in invazijo tumorskih celic, ki smo jo spremljali kontinuirano v realnem ĉasu in s spremljanjem rasti veĉceliĉnih tumorskih sferoidov. Izsledki raziskave tako razkrivajo specifiĉen mehanizem protitumornega delovanja kompleksa kliokinola z rutenijem, ki je neodvisen od splošne citotoksiĉnosti spojin. Hkrati kaţejo njegovo moţno uporabo kot spojino vodnico za razvoj novih rutenijevih spojin za terapijo raka. V okviru našega dela smo se osredotoĉili tudi na iskanje novih inhibitorjev katepsina X in preverjanje njihovega protitumornega delovanja. Z rešetanjem interne knjiţnice spojin smo kot reverzibilne in selektivne inhibitorje katepsina X identificirali spojine s triazolno strukturo. Izmed teh je imela najniţjo konstanto inhibicije spojina (1-(2,3-dihidrobenzob1,4dioksin-6-il)-2-((4-izopropil-4H-1,2,4-triazol-3-il)tio)etan-1-on) (Z9), ki je bila tudi veĉ kot stokrat bolj selektivna za katepsin X v primerjavi s sorodnimi katepsini. Spojina Z9 je znaĉilno zmanjšala migracijo tumorskih celic, poleg protitumornega delovanja pa je poveĉala tudi izrašĉanje nevritov na modelih nevrodegenerativnih obolenj. Triazolni inibitorji katepsina X so tako zanimivi kandidati za nadaljnje in vivo študije in hkrati spojine vodnice za razvoj novih inhibitorjev katepsina X.

Keywords

cisteinske proteinaze;katepsin B;katepsin X;rak (medicina);patogeneza;tumor;invazija;tumorske celice;epitelno-mezenhimski prehod;inhibitorji;disertacije;

Data

Language: Slovenian
Year of publishing:
Typology: 2.08 - Doctoral Dissertation
Organization: UL FFA - Faculty of Pharmacy
Publisher: [A. Mitrović]
UDC: 577.152.34:616-006(043.3)
COBISS: 290063872 Link will open in a new window
Views: 153
Downloads: 0
Average score: 0 (0 votes)
Metadata: JSON JSON-RDF JSON-LD TURTLE N-TRIPLES XML RDFA MICRODATA DC-XML DC-RDF RDF

Other data

Secondary language: English
Secondary title: Role of cysteine cathepsins B and X and their inhibitors in epithelial-mesenchymal transition of tumor cells
Secondary abstract: Increased expression and activity of lysosomal cysteine carboxypeptidases cathepsins B and X are associated with a variety of pathological processes, including development and progression of cancer. In cancer, they have an important role in degradation of extracellular matrix (ECM), tumor invasion, migration, metastasis and angiogenesis. In addition, epithelial-mesenchymal transition (EMT) has been recognized as an important step during tumor progression, however the role of cathepsins B and X in this process is less known. In the doctoral thesis our first goal was to investigate the involvement of cathepsins B and X in EMT and to elucidate their mechanisms of action. Using tumor cell lines differing in expression of epithelial and mesenchymal markers and cell morphology, we show that higher levels of cathepsins B and X promote EMT and are associated with mesenchymal-like cell phenotype. Additionally, a reverse process of mesenchymal to epithelial transition was triggered by their simultaneous knockdown. Among two cathepsins, cathepsin B appears to be stronger promotor of EMT. To gain insight into mechanistic role of cathepsins B and X in EMT, changes in their expression in transforming growth factor β1 (TGF-β1) signaling pathway, one of the key signaling mechanisms during EMT in cancer, were analyzed. While in epithelial breast adenocarcinoma cell line MCF7 cathepsin B expression and activity increased after triggering EMT with TGF-β1, no change in cathepsin X expression was observed. Our data thus define EMT as additional mechanism linking cathepsins B and X with tumor progression. Additionally, by silencing and inhibition of enzymatic activity tumor cells could revert to less aggressive epithelial-like cell phenotype. Cathepsins B and X are, because of their important role in cancer, validated as targets for anti-tumor therapy and are as such interesting in drug development. In the following sections of the thesis we therefore focused on identification of novel small synthetic compounds that selectively and reversibly inhibit cathepsin B and X activities and are suitable for clinical use. Cathepsin B can in addition to carboxypeptidase (exopeptidase) activity, acts as endopeptidase, due to the presence of additional structural element, termed the occluding loop. Endopeptidase activity was proposed to be predominantly responsible for the harmful cathepsin B proteolysis in cancer. In this study, using new selective synthetic inhibitors {[(8-hydroxy-5-nitroquinoline-7-yl)methyl]amino}-acetonitrile (1), 8-(4-methylpiperidin-1-yl)-5-nitroquinoline (2) and 7-[(4-methylpiperidin-1yl)methyl]-5-nitroquinolin-8-ol (3) we investigated effect of both cathepsin B activities on ECM degradation and tumor invasion. We show that endopeptidase and exopeptidase activities of cathepsin both contribute to degradation of ECM and tumor cell invasion. Taken together, this opens new strategies for regulation of its increased harmful activity by design of novel inhibitors that would simultaneously and potently impair both activities. In in vitro and in vivo tumor models, we evaluated anti-tumor properties of an established antimicrobial agent nitroxoline that was previously identified as potent and selective inhibitor of cathepsin B endopeptidase activity. On selected tumor and endothelial cell lines, expressing higher levels of active cathepsin B, nitroxoline significantly reduced extracellular DQ-collagen type IV degradation, tumor cell invasion, growth and invasion of multicellular tumor spheroids, and endothelial tube formation in the angiogenesis assay. Moreover, it significantly abrogated tumor growth, angiogenesis and metastasis in vivo on three independent tumor mouse models. Taken together, our results establish nitroxoline as promising new drug candidate for treatment of cancer. Additionally, we evaluated anti-tumor properties of the 7-aminomethylated nitroxoline derivative (compound 17). In the previous study compound 17 exhibited significantly improved kinetic properties over nitroxoline. Here we show that compound 17 more effectively reduced tumor cell invasion and migration in different cell-based models. Furthermore, it was more efficient than nitroxoline in reducing tumor growth in LPB mouse fibrosarcoma model. With the addition of 2-(ethylamino)acetonitrile group to nitroxoline, we therefore successfully improved its pharmacological properties and obtain novel candidate for anti-tumor therapy. Next, in our study, we have identified clioquinol-ruthenium complex [Ru(η6-p-cymene)(Cq)Cl] as a potent inhibitor of cathepsin B. Clioquinol-ruthenium complex did not affect cell viability in low micormolar concentrations, however it did significantly reduced degradation of ECM proteins and tumor cell invasion. Our findings for clioquinol-ruthenium complex reveal specific anti-tumor mechanism, not related to a general compound-induced toxicity, and establish it as a lead compound for development of new ruthenium based anti-tumor agents. In the final part of our work, we have focused on identifying novel cathepsin X inhibitors. By screening in-house compound library, we identified triazole-based compounds as reversible selective inhibitors of cathepsin X activity. Among them the best performing compound with the lowest constant of inhibition and at least hundred times improved selectivity for cathepsin X compared to other related cathepsins was (1-(2,3-dihydrobenzob1,4dioxin-6-yl)-2-((4-isopropyl-4H-1,2,4-triazol-3-yl)thio)ethan-1-one) (Z9). Z9 significantly reduced tumor cell migration and in addition to anti-tumor activity, it increased also the neurite outgrowth in the nevrodegeneration model. Cathepsin X inhibitors with triazole ring are therefore promising candidates for further in vivo evaluation and represent lead compounds for design of new cathepsin X inhibitors.
Type (COBISS): Dissertation
Thesis comment: Univ. v Ljubljani, Fak. za farmacijo
Pages: X, 234 str.
ID: 15654905