Identifikacija tarč katepsinov B in X v tumorskih matičnih celicah raka dojke

Kokalj, Ajda (author), Mitrović, Ana (mentor)

Pojavnost raka dojk, druge najpogostejše oblike raka pri ženskah v razvitem svetu, v zadnjih desetletjih še vedno narašča. Neuspehe pri zdravljenju raka in ponoven pojav bolezni lahko pripišemo predvsem prisotnosti tumorskih matičnih celic (TMC) – majhni subpopulaciji celic, ki je intrinzično odporna na večino obstoječih terapevtskih pristopov. Posledično glavni izziv pri razvoju novih terapevtskih strategij predstavlja učinkovito ciljanje TMC. Za sorodni lizosomski cisteinski karboksipeptidazi katepsina B in X je znano, da sta udeleženi tako v začetni stopnjah razvoja raka kot tudi v številnih kasnejših. Vpletenost v razvoj maligne bolezni ju opredeljuje kot primerni tarči za protitumorno terapijo. Za razvoj novih molekularnih orodij in uspešno uvedbo novih terapevtskih pristopov je danes ključnega pomena predvsem dobro razumevanje njihovih mehanizmov delovanja. Naš namen v tej magistrski nalogi je tako identificirati tarče katepsinov B in X v TMC raka dojk in določiti vpliv zaviralcev katepsinov B in X na molekulske mehanizme v TMC. V okviru magistrske naloge smo TMC izolirali iz treh celičnih linij raka dojk: MCF7, MCF-10A neoT in MDA-MB-231. Zaradi identifikacije tarč katepsinov B in X v TMC smo spremljali spremembe v izražanju proteinov do katerih pride po tretiranju TMC s specifičnimi nizko-molekularnimi zaviralci katepsinov in ločbi z 2D elektroforezo. Izražanje proteinov smo po ločbi detektirali z barvilom Coomassie modro in s srebrenjem. Nadalje smo določili vpliv zaviralcev katepsinov B in X v TMC na aktivnost katepsinov v celičnih lizatih in s pretočno citometrijo ovrednotili njihov vpliv na proliferacijo TMC. Pri tem smo pokazali, kateri zaviralci delujejo reverzibilno in kateri ireverzibilno, ter da uporabljeni zaviralci ne vplivajo na proliferacijo. To kaže na to, da sta katepsina B in X pri TMC udeležena preko bolj specifičnih mehanizmov. V magistrski nalogi nam tarčnih proteinov, ki jih katepsina cepita v TMC, ni uspelo identificirati, saj se je izvedba 2D elektroforeze izkazala kot precej težavna. Kljub težavam smo potrdili pomen poznavanja molekulskih mehanizmov za uvedbo novih terapevtskih pristopov in določili vpliv zaviralcev katepsinov B in X na molekulske mehanizme v TMC.

• Language: Slovenian
• Keywords: tumorske matične celice, katepsin B, katepsin X, 2D elektroforeza, zaviralci peptidaz
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2024
• PID: 20.500.12556/RUL-159201
• Publication date in RUL: 03.07.2024

Secondary language

• Language: English
• Title: Identification of cathepsin B and X targets in breast cancer stem cells

Abstract

The incidence of breast cancer, the second most common cancer in women in the developed world, has continued to rise in recent decades. Unsuccessful cancer treatments and disease remissions are mainly attributed to the presence of cancer stem cells (CSCs). CSCs are a small subpopulation of cells that are inherently resistant to most existing therapeutic approaches. A major challenge in the development of new therapeutic strategies is therefore the effective targeting of CSCs. It is already known that the related lysosomal cysteine carboxypeptidases, cathepsins B and X, play a role in both early stages and many later of cancer development. This fact makes them suitable targets for cancer therapy. Nowadays, a good understanding of the mechanisms is crucial for the development of molecular tools and the successful implementation of new therapeutic approaches. Therefore, the aim of this master thesis is to identify target proteins of cathepsin B and X in breast cancer CSCs and to determine the effects of cathepsin B and X inhibitors on the molecular mechanisms in CSCs. In the master thesis, CSCs were isolated from three breast cancer cell lines: MCF7, MCF-10A neoT and MDA-MB-231. To identify the targets of cathepsins B and X in the CSCs, we monitored the changes in protein expression that occur after treatment of CSCs with specific small molecule cathepsin inhibitors following separation by 2D electrophoresis. Protein expression was then detected by staining with Coomassie blue and silver. In addition, we determined the effect of inhibitors of cathepsins B and X in CSCs, on the activity of cathepsins in cell lysates and evaluated their effect on the proliferation of CSCs by flow cytometry. We were able to show which inhibitors act reversibly and which irreversibly, and that the inhibitors we used do not affect cell proliferation. This suggests that cathepsins B and X are involved in CSCs via more specific mechanisms. In the master thesis, we were not able to identify the targets cleaved by the cathepsins in CSCs as 2D electrophoresis proved to be quite difficult. However, we confirmed the importance of knowing the molecular mechanisms for the introduction of new therapeutic approaches and determined the influence of cathepsin B and X inhibitors on the molecular mechanism in CSCs.

• Keywords: cancer stem cells, cathepsin B, cathepsin X, 2D electrophoresis, peptidase inhibitors