Vrednotenje citotoksičnih učinkov zaviralcev proteasoma v odvisnosti od izražanja njegovih podenot β5

Sever, Ana (author), Gobec, Martina (mentor), Šmid, Alenka (comentor)

Proteasom in imunoproteasom sta pomembna encimska kompleksa v človeškem telesu. Prvi je prisoten v vseh celicah, drugi pa predvsem v celicah imunskega sistema, kjer ima pomembno vlogo v mnogih procesih. Zato je zanimiva tarča pri zdravljenju številnih avtoimunskih bolezni in rakavih obolenj. Med njimi je tudi kronična limfocitna levkemija, ki kljub razvoju in napredku terapije v zadnjih letih še vedno ostaja neozdravljiva. V ta namen se iščejo nove tarče, ki bi lahko vodile v njeno uspešnejše zdravljenje. Med raziskovalnim delom smo najprej opazovali vlogo proteasoma in imunoproteasoma na izoliranih limfocitih B 11 pacientov z diagnosticirano KLL. Spremljali smo in vitro citotoksičnost neselektivnega zaviralca karfilzomiba in dveh selektivnih zaviralcev imunoproteasoma PR-957 in DPLG-3. Citotoksičnost smo merili s testom metabolne aktivnosti po 24 in 48 urah, s čimer smo določili srednjo zaviralno koncentracijo (IC50). Pokazali smo, da so vsi trije zaviralci citotoksični na celice KLL in vitro, vendar vsak v svojem koncentracijskem območju. Najbolj citotoksičen je karfilzomib (IC50=2,95 nM), sledi mu PR-957 (IC50=40,62 nM), najmanj citotoksičen pa je DPLG-3 (IC50=2461 nM). Citotoksičnost je tudi časovno odvisna – po 48 urah so IC50 vrednosti za 85% nižje. V nadaljevanju smo s pomočjo kvantitativne verižne reakcije s polimerazo v realnem času določali izražanje genov za podenote proteasoma in imunoproteasoma (β5c, β5i, β2c, β2i, β1c, β1i). Zanimalo nas je predvsem izražanje podenot β5c in β5i, saj se zaviralci, s katerimi smo preverjali citotoksičnost, v različni meri selektivno vežejo na ti dve podenoti. Ugotovili smo, da le eden izmed izbranih vzorcev izraža podenoto β5i bolj kot β5c. Korelacije med citotoksičnostjo zaviralcev in genskim izražanjem posameznih podenot nismo ugotovili. Za konec smo želeli preveriti še izražanje podenot proteasoma in imunoproteasoma na ravni proteinov. Za določanje le-teh smo proteine prenesli na membrano s postopkom western in želene podenote določili s pomočjo specifičnih protiteles. Zanimalo nas je, če gensko izražanje podenot korelira z izražanjem podenot na ravni proteinov. Te povezave nismo ugotovili, zato smo dodatno preverili, če izražanje podenot na ravni proteinov korelira s citotoksičnim učinkom zaviralcev. Tudi te hipoteze nismo mogli potrditi. Statistično značilne korelacije med različnimi parametri nismo uspeli potrditi. Eden izmed razlogov je lahko tudi prenizko število vzorcev, na katerih smo opravljali raziskavo, zato bi bilo v prihodnje potrebno povečati njihov nabor in ponoviti analize.

• Language: Slovenian
• Keywords: kronična limfocitna levkemija, imunoproteasom, zaviralci imunoproteasoma
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2019
• PID: 20.500.12556/RUL-112727
• Publication date in RUL: 08.11.2019

Secondary language

• Language: English
• Title: Evaluation of proteasome inhibitors cytotoxic effects in correlation with the expression of its β5 subunits

Abstract

ABSTRACT Proteasome and immunoproteasome are two important enzyme complexes in our body. The first one is expressed in all cells of the body, while the other plays a pivotal role in the cells of the immune system. Deregulation of the immunoproteasome is associated with some diseases, thus it presents itself as an attractive therapeutic target, especially in autoimmune diseases and different types of cancer. Chronic lymphocytic leukemia (CLL), which despite a lot of research and quite a few existing treatments, remains incurable. With that in mind, there is an ongoing search to find new therapeutic agents to optimize the current therapy. In the course of study, the role of proteasome and immunoproteasome inhibition on 11 samples of lymphocytes B obtained from patients with diagnosed CLL, was being observed. In vitro cytotoxicity of non-selective inhibitor carfilzomib and two selective inhibitors PR-957 and DPLG-3 was determined. Cytotoxicity was evaluated using the metabolic activity of cells at 24 and 48 hours. Mean inhibitory concentration (IC50) was calculated and revealed that all of the inhibitors are cytotoxic to CLL cells, but each in their respective concentration range. Carfilzomib was the most cytotoxic (IC50=2,95 nM), followed by PR-957 (IC50=40,62 nM) and DPLG-3 as the least cytotoxic (IC50=2461 nM). Cytotoxicity was also time dependent, with IC50 values dropping for 85% at the 48 hour mark. Next, gene expression of proteasome and immunoproteasome subunits (β5c, β5i, β2c, β2i, β1c, β1i) was evaluated using quantitative real time polymerase chain reaction. The main focus was on subunits β5c and β5i, which are the main targets of the chosen inhibitors. Only one of the CLL samples expressed the β5i subunit higher than β5c. No correlation between the cytotoxicity of inhibitors and the level of gene expression of individual subunits could be determined. In the end, we addressed the protein expression of proteasome and immunoproteasome subunits (β5c, β5i, β2c, β2i, β1c, β1i) with the use of western blot method and specific antibodies. Statistical analysis did not reveal a correlation between the gene and protein subunit expression. Moreover, no correlation between protein subunit expression and cytotoxicity of inhibitors could be demonstrated. Although no statistically significant correlations were determined, further studies with more samples will be necessary to provide a better and more relevant understanding of the topic.

• Keywords: chronic lymphocytic leukemia, immunoproteasome, immunoproteasome inhibitors