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.
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