Vrednotenje proteinskih nivojev in aktivnosti katalitičnih podenot proteasomov v izbranih celičnih linijah raka dojke

Motaln, Mirjam (avtor), Gobec, Martina (mentor), Smrdel, Lara (komentor)

Proteasom je velik proteinski kompleks, odgovoren za proteolizo znotrajceličnih proteinov. Hematopoetske celice izražajo specializirano obliko proteasomov, znanih kot imunoproteasomi, v katerih so konstitutivne katalitično aktivne podenote ß1, ß2, ß5 nadomeščene z induciranimi homologi ß1i, ß2i in ß5i. Ker je proteasom dobro znana tarča pri rakavih obolenjih hematološkega izvora, smo skušali raziskati, ali raven katalitičnih podenot proteasomov vpliva na občutljivost izbranih celičnih linij raka dojke na citotoksične učinke zaviralcev proteasomov. Izbrali smo celične linije raka dojke z različnim metastatskim potencialom in jih tretirali z IFN-γ, ki poviša izražanje imunoproteasoma, v koncentracijsko in časovno odvisnem režimu. S prenosom western in uporabo sond za določanje aktivnosti encimskih podenot proteasomov smo dokazali, da izpostavljenost celičnih linij koncentraciji IU/mL IFN-γ spodbudi izražanje podenot β1i, β2i in β5i, ki je najbolj izrazito po 48 urah. Nato smo proučili še citotoksične učinke zaviralcev proteasomov (bortezomib, karfilzomib, KZR-504, LMP7-IN-1) na izbrane celične linije s testom metabolne aktivnosti. Ugotovili smo, da se citotoksični profili izbranih zaviralcev v odsotnosti ali prisotnosti IFN-γ bistveno ne spremenijo. V nadaljevanju smo se osredotočili na nivo izražanja proteasomov v rakavih celičnih linijah in morebitnega vpliva na mikrookolje, ki nastane ob interakciji s trombociti. Uporabili smo kokulture trombocitov in celic raka dojke, saj takšni modeli in vitro bolje posnemajo pogoje in vivo v smislu imunosupresivnega mikrookolja, ki omogoča nastanek metastaz. Rakave celice smo predhodno tretirali z IFN-γ ali zaviralci proteasoma, nato pa tvorili kokulture z izoliranimi trombociti. S pretočno citometrijo smo določali ravni izločanja izbranih kemokinov (IL-8, CCL2, CCL5, MIG, CXCL9, CXCL10). Analiza supernatantov je pokazala velike razlike v ravneh izločanja kemokinov CCL5 in IL-8 napram bazalnemu izločanju iz posameznih celic. Opazili smo tudi spremembe pri ravneh izločenega CXCL10 in MIG po tretiranju z zaviralci proteasoma, kar kaže, da na proces degranulacije lahko vplivamo s tretiranjem celic s selektivnimi zaviralci proteasomov. Rezultati nakazujejo na morebitno vlogo proteasoma na tumorsko mikrookolje, a so potrebne nadaljnje študije, da bi ugotovili, ali bi proteasom lahko služil kot potencialna tarča za zdravljenje metastazirajočega raka dojke.

• Jezik: Slovenski jezik
• Ključne besede: proteasom, rak dojke, zaviralci proteasoma, trombociti
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2023
• PID: 20.500.12556/RUL-151093
• Datum objave v RUL: 29.09.2023

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Determination of protein levels and catalytic activities of proteasome subunits in selected breast cancer cell lines

Izvleček

Proteasome is a large protein complex responsible for intracellular proteolysis. Hematopoietic cells assemble a specialized form of proteasomes, known as the immunoproteasomes, in which the constitutive catalytic sites ß1, ß2, ß5 are replaced by the inducible homologs ß1i, ß2i and ß5i. As proteasome is a well-known target in haematological malignancies, we sought to investigate whether the level of catalytic subunits of proteasomes affects the susceptibility of breast cancer cells to the cytotoxic effects of proteasome inhibitors. Breast cancer cell lines with different metastatic potential were treated with IFN-γ that up-regulates the immunoproteasome expression in time- and dose-dependent matter. By western blotting and activity profiling with irreversible fluorescent probes, we determined that exposure of cell lines to a 150 IU/mL concentration of IFN-γ stimulates the expression of β1i, β2i and β5i immunoproteasome subunits which is most noticeable after 48 hours. Subsequently, the cytotoxic effect of proteasome inhibitor treatment (bortezomib, carfilzomib, KZR-504, LMP7-IN-1) was determined by a metabolic activity assay. We determined that presence or absence of IFN-γ has no significant impact on cytotoxic profiles of used proteasome inhibitors We next used co-cultures of platelets and breast cancer cells, as such in vitro models better mimic the in vivo conditions in terms of the immunosuppressive microenvironment that enables the formation of metastases. Breast cancer cells were pre-treated with IFN-γ or proteasome inhibitors and later co-cultured with isolated platelets. By flow cytometry levels of selected chemokines (IL-8, CCL2, CCL5, MIG, CXCL9, CXCL10) were assessed. Analysis of the supernatants showed large differences in the levels of CCL5 and IL-8 secretion compared to the basal secretion from the cells. We also observed significant changes in the levels of secreted CXCL10 and MIG after treatment with proteasome inhibitors, which indicates that the degranulation process can be influenced by treating cells with selective proteasome inhibitors. These results indicate the potential role of proteasome in shaping the tumour microenvironment. However, further studies are required to ascertain whether proteasome could serve as an efective therapeutic against metastatic breast cancer.

• Ključne besede: proteasome, breast cancer, proteasome inhibitors, platelets