20.500.12556/RUL-170843 Preučevanje udeleženosti katepsinov B in X v izbranih signalnih poteh v tumorskih matičnih celicah raka dojke Investigating the involvement of cathepsins B and X in selected signaling pathways in cancer stem cells of breast cancer V magistrskem delu smo preučevali udeleženost dveh lizosomskih cisteinskih peptidaz, katepsinov B in X, v kanonični signalni poti Wnt in s kalcijem povezani signalni poti v tumorskih matičnih celicah (TMC) raka dojke. Za ta namen smo TMC izolirali iz treh celičnih linij raka dojke MDA-MB-231, MCF7 in MCF-10A neoT na podlagi njihove sposobnosti tvorbe tumorskih sfer. V izoliranih TMC smo zavirali aktivnost katepsinov B in X z uporabo nizko molekularnih reverzibilnih zaviralcev katepsina B (nitroksolin) in katepsina X (spojina Z9) ali njuno kombinacijo. V celičnih lizatih smo po ločbi proteinov z metodo NaDS-PAGE, prenosu western in detekciji z uporabo specifičnih primarnih in sekundarnih protiteles ovrednotili vpliv zaviranja katepsinov B in X na relativno količino β-katenina, SOX2, ciklina D1, CD44, c-Myc in DCLK1 v sklopu signalne poti Wnt in kalumenina v sklopu s kalcijem povezane signalne poti. Lokalizacijo β-katenina smo v celičnih lizatih dodatno preverili po frakcionaciji celic in s konfokalno fluorescenčno mikroskopijo. Zaviranje katepsinov B in X je vplivalo na relativno količino analiziranih proteinov, kar nakazuje, da sta katepsina B in X verjetno udeležena v regulaciji kanonične signalne poti Wnt in s kalcijem povezane signalne poti. Pri tem se je ob sočasnem zaviranju katepsinov B in X v TMC celične linije MCF-10A neoT statistično značilno povečala količina β-katenina v jedru TMC. Pri ostalih proteinih je prišlo do povečanja ali zmanjšanja količine, pri čemer je bil vpliv zaviranja katepsinov B in X različen pri različnih proteinih in pri TMC različnih celičnih linij raka dojke. In this master thesis, we were investigating the involvement of two lysosomal cysteine peptidases, cathepsins B and X, in canonical Wnt signaling pathway and calcium-related signaling pathway in cancer stem cells (CSCs) of breast cancer. For this purpose, we isolated CSCs from the three breast cancer cell lines MDA-MB-231, MCF7 and MCF-10A neoT based on their ability to form tumorspheres. In the isolated CSCs the activity of cathepsins B and X was inhibited using small molecule reversible inhibitors of cathepsin B (nitroxoline) and cathepsin X (compound Z9) or their combination. In cell lysates, the effect of inhibition of cathepsins B and X on the relative quantity of β-catenin, SOX2, cyclin D1, CD44, c-Myc and DCLK1 in the Wnt signaling pathway and calumenin in the calcium-related signaling pathway was evaluated after protein separation by SDS-PAGE, western blot and detection with specific primary and secondary antibodies. The localisation of β-catenin in cell lysates was further analysed by cell fractionation and confocal fluorescence microscopy. Inhibition of cathepsins B and X affected the relative quantity of the analysed proteins, suggesting that cathepsins B and X are probably involved in the regulation of the canonical Wnt signaling pathway and the calcium-related signaling pathway. Simultaneous inhibition of cathepsins B and X in the CSCs of the MCF-10A neoT cell line resulted in a statistically significant increase in the quantity of β-catenin in the nucleus of CSCs. For the other proteins, there was an increase or decrease in protein quantity, although the effect of inhibition of cathepsins B and X was different for different proteins and in CSCs of different breast cancer cell lines. rak dojke tumorske matične celice katepsin B katepsin X signalne poti breast cancer cancer stem cells cathepsin B cathepsin X signaling pathways true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2025-07-18 07:15:54 2025-07-18 07:16:00 2025-07-20 03:44:32 0000-00-00 00:00:00 2025 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 257840 243032835 49479.pdf 49479.pdf 1 0C0AD2E9DFAC39F4B31EB89619594850 463c34260afa56df1a1a90a77dbfd2bae3e4b8a17da8513d78ac9801ecfb3eec 4adfc1a3-6396-11f0-9328-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=213102 Biotehniška fakulteta 0 0 0