Vpliv zaviralcev katepsina X na živost in migracijo celic raka prostate PC-3

Bohinc, Klara (author), Pečar Fonović, Urša (mentor)

Katepsin X je cisteinska peptidaza, vključena v potek različnih bolezni. Fiziološko je njegovo nahajanje omejeno skoraj izključno na celice imunskega sistema, zaznali pa so ga tudi že v pljučih in centralnem živčnem sistemu. Ob prekomernem izražanju in višji proteolitski aktivnosti, s cepitvijo svojih molekularnih tarč, vplivom na signaliziranje in uravnavanjem delovanja imunskega sistema, močno vpliva na potek in prognozo rakavih obolenj ter vnetnih in nevnetnih nevrodegenerativnih bolezni. Ker endogeni zaviralci nanj ne delujejo, so se raziskave obrnile v smer eksogenih. To so bodisi spojine z ireverzibilno naravo zaviranja in nizko selektivnostjo bodisi reverzibilne narave z večjo selektivnostjo, a ne dovolj zadovoljivo učinkovitostjo. V okviru diplomske naloge smo testirali pet triazolnih zaviralcev katepsina X: CX24, CX26, CX32, CX38 ter CX14 F2-8. Ti so bili sintetizirani na podlagi strukture zaviralca Z9 in imajo primerljivo vrednost 〖IC〗_50. V prvem delu diplomske naloge smo na celični liniji raka prostate PC-3 s testom MTS preverili njihov vpliv na preživetje celic po 24-urni in 48-urni inkubaciji. V koncentraciji 10 M sta od petih zaviralcev v obeh časovnih obdobjih šibko citotoksičnost izkazala CX24 ter CX14 F2-8. Živost je po 24-urni inkubaciji v primeru CX24 znašala 87 %, po 48-urni pa 90 %. V primeru CX14 F2-8 je po 24 urah preživelo 93 % celic, po 48 urah pa 91 %.V primeru CX32 in CX38 do statistične razlike v preživetju v primerjavi s kontrolo ni prišlo, tako da smo ju določili za necitotoksična, zaviralcu CX26 pa smo po 48-urni inkubaciji, kljub visoki živosti (96 %), izračunali statistično značilno zmanjšanje preživetja. Zaradi slednjega in dejstva, da ima vrednost 〖IC〗_(50 ) rahlo slabšo od starševske spojine Z9, smo v nadaljnje testiranje prenesli samo zaviralca CX32 in 38. Vpliv zaviralcev na migracijo celic smo s pomočjo sistema xCELLigence testirali v isti koncentraciji, neprekinjeno 72 ur. Nobeden od izbranih zaviralcev ni zavrl migracije, sta jo pa, v nasprotju s pričakovanji, povečala. Testirane spojine smo v okviru diplomske naloge sicer dokazali kot necitotoksične ali šibko citotoksične na celicah raka prostate PC-3, a hkrati neučinkovite pri zmanjšanju njihove migracije. Njihova struktura lahko služi za nadaljnji razvoj spojin, primernih za zaviranje katepsina X. Ker ima le-ta vpliv tudi na vnetno in nevnetno nevrodegeneracijo, bi predlagali testiranje novih specifičnih zaviralcev tudi na tem področju.

• Language: Slovenian
• Keywords: katepsin X, selektivni zaviralci, test MTS, migracija
• Work type: Bachelor thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2020
• PID: 20.500.12556/RUL-119632
• Publication date in RUL: 10.09.2020

Secondary language

• Language: English
• Title: The influence of cathepsin X inhibitors on viability and migration of PC-3 prostate cancer cells

Abstract

Cathepsin X is a cysteine peptidase involved in the development of various diseases. Physiologically, it is located almost exclusively in the cells of the immune system, but has also been detected in the lungs and the central nervous system. During its excessive expression and proteolytic activity, with cleavage of its molecular targets, influence on the signalisation and the modulation of the immune system, cathepsin X strongly affects the course and the prognosis of malignancies, as well as inflammatory or non-inflammatory neurodegenerative diseases. Due to the ineffectiveness of endogenous inhibitors, the research now focuses on exogenous inhibitors. These compounds are either irreversible with low selectivity or reversible with higher selectivity, but insufficient effectiveness. In this thesis, we tested five triazole-based cathepsin X inhibitors: CX24, CX26, CX32, CX38, and CX14 F2-8. These were synthesized based on the structure of inhibitor Z9 and have comparable 〖IC〗_50 values. First, we used the MTS assay to analyse their influence on cell viability on the PC-3 prostate cancer cell line after 24- and 48-hour incubation. After both time periods, two inhibitors, CX24 and CX14 F2-8, displayed weak cytotoxic effects in the 10 M concentration. The viability of CX24 was 87 % after 24 and 90 % after 48 hours. With CX14 F2-8, 93 % of cells survived the 24- and 91 % the 48-hour period. CX32 and CX38 were marked as non-cytotoxic based on no statistically significant difference in cell survival between the tested compounds and positive control. In spite of the 96 % viability rate, we calculated a statistically significant decrease for inhibitor CX26. Due to these results and its slightly inferior 〖IC〗_(50 )value to that of the parent compound Z9, only CX32 and CX38 were included in further testing. Using the same concentration as before, we tested their influence on cell migration with the xCELLigence system, uninterruptedly for 72 hours. Contrary to expectations, the compounds did not inhibit, but increased the migration. In the thesis, we proved the compounds to be either non-cytotoxic or to have weak cytotoxic effects on PC-3 prostate cancer cell line. However, they did not lower their migration rate. Their structure could be useful for further development of compounds suitable for cathepsin X inhibition. Because of its impact on inflammatory and non-inflammatory neurodegeneration, we would suggest testing of new specific inhibitors in this area as well.

• Keywords: cathepsin X, selective inhibitor, MTS assay, migration