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Izolacija in karakterizacija tumorskih matičnih celic iz celičnih linij raka dojke in ovrednotenje izražanja katepsinov B in X v njih
ID Zupančič, Urban (Author), ID Kos, Janko (Mentor) More about this mentor... This link opens in a new window, ID Mitrović, Ana (Co-mentor)

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Abstract
V sodobni medicini je znanih veliko zdravil in terapevtskih pristopov, ki so namenjeni zdravljenju rakavih obolenj. Kljub temu pa se bolezen pogosto razvije v invazivno obliko, ki metastazira, ali pa pride do ponovnega pojava bolezni. Za omenjene procese naj bi bile odgovorne tumorske matične celice (TMC), manjša podskupina rakavih celic, ki je odporna na večino obstoječih terapevtskih pristopov in omogoča ponovni pojav tumorjev. Za izboljšanje terapije so tako potrebni novi terapevtski pristopi, ki bi bili poleg diferenciranih tumorskih celic učinkoviti tudi proti TMC. Obetavni tarči, z regulacijo katerih bi lahko izboljšali terapijo usmerjeno proti TMC, sta cisteinski lizosomski peptidazi katepsina B in X, saj imata pomembno vlogo v različnih procesih nastanka in napredovanja raka. V okviru magistrske naloge smo TMC izolirali iz treh celičnih linij raka dojke, MDA-MB-231, MCF7 in MCF-10A neoT, na podlagi tvorbe tumorskih sfer, kar je značilnost, po kateri se TMC razlikujejo od diferenciranih celic. V ta namen smo najprej izbrali optimalne pogoje za tvorbo tumorskih sfer. Kot najbolj ugodno se je izkazalo gojišče DMEM/F-12 z brezserumskim dodatkom za spodbujanje rasti TMC B-27 in rastnih faktorjev EGF in bFGF. Pri celicah MCF-10A neoT pa je potreben še dodatek inzulina in hidorkortizona. Uspešnost izolacije TMC smo potrdili na podlagi izražanja površinskih označevalcev in jedrnega transkripcijskega dejavnika matičnih celic SOX2. Delež celic s fenotipom TMC se je pri celicah, izoliranih po tvorbi tumorskih sfer, pri vseh uporabljenih celičnih linijah povečal, kar je pokazalo povečano izražanje površinskih označevalcev (CD44+/CD24–) in/ali povečano izražanje SOX2. Nadalje smo s prenosom western, testom ELISA in z uporabo encimske kinetike pokazali, da se pri TMC iz vseh uporabljenih celičnih linij značilno povečata izražanje in aktivnost katepsina B v primerjavi z diferenciranimi celicami. Podobno sta se povečala tudi izražanje in aktivnost katepsina X pri TMC iz celičnih linij MDA-MB-231 in MCF7, medtem ko je pri TMC MCF-10A neoT prišlo le do povečanega izražanja katepsina X, ne pa tudi njegove povečane aktivnosti. Dobljeni rezultati tako odpirajo spodbudno možnost, da bi z zaviranjem katepsinov B in X v TMC lahko povečali učinkovitost protitumornih terapij proti TMC in izboljšali zdravljenje raka.

Language:Slovenian
Keywords:rak, tumorske matične celice, katepsin B, katepsin X
Work type:Master's thesis/paper
Organization:FFA - Faculty of Pharmacy
Year:2020
PID:20.500.12556/RUL-114380 This link opens in a new window
Publication date in RUL:25.02.2020
Views:1706
Downloads:337
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Secondary language

Language:English
Title:Isolation and characterization of cancer stem cells from breast cancer cell lines and evaluation of their cathepsin B and X expressions
Abstract:
In modern medicine many drugs and therapeutic approaches are available for the treatment of cancer. However, the disease often develops into an invasive metastatic form or recurs after years of remission. This may be attributed to cancer stem cells (CSCs), a small subset of cells with a tumorigenic potential that is resistant to most of therapeutic approaches. Thus, new therapeutic approaches effective, not only towards differentiated cancer cells, but also CSCs, are required. Lysosomal cysteine peptidases, cathepsin B and X that have important role in different processes of development and progression of cancer, could serve as promising molecular targets to improve CSCs-directed therapy. First, we isolated cancer stem cells from three breast cancer cell lines MDA-MB-231, MCF7 and MCF-10A neoT, based on their ability to form tumorspheres, a characteristic that distinguishes CSCs from differentiated cells. For this purpose, we first determined the optimal conditions for tumorsphere formation. The most optimal medium for tumorsphere formation was DMEM/F-12 with serum free supplement for CSC growth B-27, and growth factors EGF and bFGF. Additionally, insulin and hydrocortisone were added for growth of MCF-10A neoT cells. Isolation of CSCs was confirmed by expression of cell surface markers and stem cell transcription factor SOX2. Following tumorsphere formation surface markers (CD44+/CD24–) and/or increased expression of SOX2 showed increase in cells with CSC phenotype. Next, we demonstrated by western blot, ELISA and enzyme kinetics assay that protein levels and activity of cathepsin B were significantly increased in CSCs following tumorsphere formation compared to differentiated cells from all three cells lines. Similarly, protein levels and activity of cathepsin X were increased in CSCs from MDA-MB-231 and MCF7 cells, while CSCs from MCF-10A neoT cells showed only increased protein levels of cathepsin X, but not its activity. Taken together, we showed that inhibition of cathepsin B and X in CSCs, using specific inhibitors, is a promising approach to increase the effectiveness of current antitumor therapies against CSCs and therefore to improve cancer treatment.

Keywords:cancer, cancer stem cells, cathepsin B, cathepsin X

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